Articles in PresS. Physiol Genomics (April 27, 2010). doi:10.1152/physiolgenomics.00033.2010
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Pathogenic peptide deviations support a model of adaptive evolution of chordate cardiac
performance by troponin mutations
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Department of Integrative Biology and Physiologya and the Center for Drug Designb, University
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of Minnesota Academic Health Center, Minneapolis, Minnesota 55455, USA; Antiviral Research
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Center, Department of Medicine, University of California, San Diego, San Diego, California
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92103, USAc; Montreal Neurological Institute and Department of Biology, McGill University,
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Montreal, Quebec, Canadad; Departments of Computer Science and Physics, Virginia
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Polytechnic Institute and State University, Blacksburg, VA 24061, USAe
Nathan J. Palpanta, Evelyne M. Houangb, Wayne Delportc, Kenneth E.M. Hastingsd, Alexey V.
Onufrieve, Yuk Y. Shamb, and Joseph M. Metzgera
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Correspondence directed to:
Joseph M. Metzger Ph.D.
Department of Integrative Biology & Physiology
University of Minnesota Medical School
6-125 Jackson Hall
321 Church Street SE
Minneapolis, MN 55455
phone 612.625.5902
fax 612.625.5149
email: [email protected]
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Research Article
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Running head: Molecular evolution of cardiac troponin
Key words: myofilament, mammal, amphibian, histidine, single nucleotide polymorphism
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Copyright © 2010 by the American Physiological Society.
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Abstract
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In cardiac muscle, the troponin (cTn) complex is a key regulator of myofilament calcium
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sensitivity because it serves as a molecular switch required for translating myocyte calcium
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fluxes into sarcomeric contraction and relaxation. Studies of several species suggest that
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ectotherm chordates have myofilaments with heightened calcium responsiveness. However,
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genetic polymorphisms in cTn that cause increased myofilament sensitivity to activating calcium
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in mammals result in cardiac disease including arrhythmias, diastolic dysfunction, and increased
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susceptibility to sudden cardiac death. We hypothesized that specific residue modifications in
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the regulatory arm of troponin I were critical in mediating the observed decrease in myofilament
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calcium sensitivity within the mammalian taxa. Methods and Principle Findings: We
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performed large scale phylogenetic analysis, atomic resolution molecular dynamics simulations
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and modeling, and computational alanine scanning. This study provides evidence that this His to
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Ala substitution within mammalian cTnI reduced the thermodynamic potential at the interface
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between cTnI and cTnC in the calcium saturated state by disrupting a strong intermolecular
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electrostatic interaction. This key residue modification reduced myofilament calcium sensitivity
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by making cTnI molecularly untethered from cTnC. Conclusion: In order to meet the
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requirements for refined mammalian adult cardiac performance, we propose that compensatory
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evolutionary pressures favored mutations that enhanced the relaxation properties of cardiac
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troponin by decreasing its sensitivity to activating calcium.
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Introduction
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Evolutionary pressures favor mutations that mitigate the susceptibility to disease,
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especially as it pertains to the reproductive fitness of an organism(5, 48). Consequently,
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monogenic diseases provide excellent reference points from which to understand the
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implications of certain changes as a retrospective view on evolutionary modifications(5, 48). In
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other words, human disease resulting from pathogenic peptide deviations, defined here as single
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nucleotide polymorphisms (SNPs) that cause pathologies recapitulating non-mammalian
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physiology, provide insight into the influence of selective pressures on specific residues or
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domains of a given protein to accommodate highly specified physiological requirements(5, 48,
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54). This study assessed phylogenetic changes that occurred during evolution of mammalian
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myofilament calcium sensitivity with a focus on troponin, the regulatory molecular switch
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complex of the myofilaments.
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Troponin is a heterotrimeric protein complex comprised of three subunits. These include
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the Tm binding subunit, troponin T (TnT), the calcium binding subunit, troponin C (TnC), and
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the actomyosin ATPase inhibitory subunit, troponin I (TnI) (Figure 1a). The coordinated actions
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of the troponin complex are specifically designed to regulate the functions of acto-myosin cross-
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bridges in a calcium-dependent manner during the rhythmic transitions between contraction
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(systole) and relaxation (diastole) (38).
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During systole, myofilament activation is mediated through binding of the TnI switch
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arm to an exposed hydrophobic patch on the calcium-bound N-terminal domain of cTnC(4, 41,
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45). These actions enable coordination of the azimuthal positioning of Tm in the actin groove by
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TnT and subsequent sarcomeric contraction mediated by actin-myosin cross-bridge cycling (38,
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66, 100). The biophysics of myofilament contraction are translated into ventricular inotropic
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performance at the whole organ level(38). During diastole, calcium is released from cTnC and
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sequestered into the SR allowing the regulatory arm of TnI to translocate into an inhibitory
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position on actin. This is sufficient to inhibit actin-myosin cross bridge activation resulting in
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sarcomeric relaxation(60, 71). Förster resonance energy transfer (FRET) experiments have
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shown that the movements of the regulatory arm of TnI (TnIreg) during systole and diastole are
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essential for the transmission of the calcium signal to the rest of the myofilament proteins(60,
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71).
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The specific amino acid composition of the cTn complex markedly affects its sensitivity
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to activating calcium which, ultimately, influences contraction (inotropic) and relaxation
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(lusitropic) performance(20, 21, 33, 37, 92, 112). Compared to mammals, studies of several
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species suggest that ectotherms such as fish and amphibians require heightened myofilament
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calcium sensitivity to maintain cardiac performance in the context of challenging environmental
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conditions such as cold temperatures and extended periods of hypoxia(32, 34, 35, 61, 62, 88, 89).
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However, SNPs in cTn that cause increased myofilament sensitivity to activating calcium in
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mammals, termed here pathogenic peptide deviations, result in cardiac disease including
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arrhythmias, diastolic dysfunction, and increased susceptibility to sudden cardiac death(9, 20, 72,
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101). These clinical observations reveal an absolute requirement for reduced myofilament
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sensitivity to activating calcium within the mammalian taxa compared to other phylogenies.
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Using large scale bioinformatics and an array of molecular modeling techniques we propose that
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the intrinsic relaxation potential of cardiac troponin has been enhanced during chordate evolution
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to meet the functional requirements of the mammalian cardiovascular system.
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Methods
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Amino acid and mRNA sequences and alignments Sequences were extracted by search of
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NCBI protein and nucleotide databases as well as the UniProt KB protein database. Defining the
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taxanomic relationship between species studied was accomplished by use of the NCBI taxonomy
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database (Supplemental Figure 1). Table 1 outlines abbreviations used to reference specific
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species. Supplemental Table 1 outlines all relevant details on the sequences acquired from NCBI
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or UniProt KB databases used in this phylogenetic analysis. To be consistent with prior studies,
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all amino acid numbering in this study refers to the adult rat isoform sequence (cTnI, cTnC, and
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cTnT) including the starting methionine unless otherwise stated. Protein and nucleotide
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sequences were aligned by Clustal W analysis as originally described by Thompson et al(99).
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Multiple alignment parameters were as follows: gap penalty = 15; gap length penalty = 6.66;
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protein weight matrix: Gonnet Series. Pairwise analysis of percent identity between sequences
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was used to define the relationship between proteins or within proteins across designated species.
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Genome scans for novel TnI genes New sequences for troponin I isoforms were acquired by
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whole genome scans of numerous species. Genome BLAST analysis for TnI isoforms were
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performed on Anolis carolinensis (lizard, USCS), Taeniopygia guttata (zebra finch, NCBI),
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Cavia porcellus (guinea pig, UCSC), and Callithrix jacchus (marmoset, UCSC). Query
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sequences from taxonomically related species were used for the genome scans. Predicted isoform
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for TnI sequences was accomplished by Clustal W analysis followed by sequence percent
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identity and phylogenetic tree analysis. The Kimura distance formula was used to calculate
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distance values, derived from the number of non-gap mismatches and corrected for silent
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substitutions. The values computed are the mean number of differences per site and fall between
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0-1. Zero represents complete identity and 1 no identity. Scale of the phylogenetic tree indicates
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the number of nucleotide substitutions per 100 residues. Phylogenetic tree analysis was used to
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confirm identity of predicted TnI isoforms by sequence correlation with other TnI isoforms.
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Sequence ID and divergence values were calculated to define the relationship between novel TnI
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proteins and related TnI isoforms to assist in confirmation of postulated isoform designation.
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Divergence (i,j) is calculated as (100[Distance (i,j)] / Total Distance) where (i,j) is the sum of the
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branch lengths between two sequences and Total Distance is the sum of all branch lengths. All
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data regarding protein and mRNA sequences are outlined in Supplemental Table 2, and
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Supplemental Figures 2 (nucleotide) and 3 (protein).
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Determination of evolutionary selective pressure Phylogenetic codon models were used to
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understand the selective pressure on the both the full gene sequence of cardiac TnI and on the
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switch domain. Briefly, codon-based methods (recently reviewed by (2, 24)) make use of a
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continuous-time Markov process to model synonymous (dN) and non-synonymous substitutions
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(dS) along a phylogenetic tree. Phylogenetic trees were estimated, using PHYML (40),
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independently for the full gene sequence and for the switch domain, since only a subset of full
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cTnI sequences were available for the mammalian taxa of interest. The switch domain is short
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(63 nucleotides), and thus some internal branches of the phylogeny had lengths of zero; we
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collapsed those branches into polytomies. It was assumed that synonymous and non-synonymous
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substitution rates at codon sites can be approximated using discrete distributions, the parameters
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of which are estimated by maximum likelihood. To rule out the potential confounding effect of
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recombination or gene conversion, the alignment was screened using GARD(51, 52); no
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evidence for recombination was detected. To identify the distribution which best describes
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synonymous and non-synonymous rate variation across all sites a step-wise procedure was used
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starting at a single rate for all sites and progressively adding new rate classes, each time
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estimating the synonymous and non-synonymous rates of each class and evaluating model fit
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using the small sample correction to the Akaike’s Information Criterion (AIC-c)(93). The
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stepwise iteration was terminated once no further improvement in model fit was achieved. A
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Fixed Effects Likelihood (FEL) methods (implemented on www.datamonkey.org(80)) was used
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to estimate the synonymous to non-synonymous substitution rate at each site independently for
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the switch domain. Briefly, evidence for either positive (dN/dS > 1) or purifying selection
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(dN/dS < 1) was identified by comparing the fit of a model in which synonymous and non-
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synonymous rates are constrained to be equal, to that in which this constraint is relaxed.
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Statistical significance was evaluated with a Likelihood Ratio Test (P < 0.05).
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Molecular modeling and structural analysis All modeling was carried out using Maestro
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version 9.0.111 (Schrödinger, LLC, New York, NY; 2007). The solved X-ray crystallographic
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structures of human cardiac troponin(95) (PDB: 1J1E, 3.3 Å) and skeletal troponin(103) (PDB:
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1YTZ, 3.0 Å) in complex with Ca++ bound troponin C were taken from the Protein Data Bank.
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The missing hydrogen atoms were added to both X-ray structures followed by energy
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minimization using OPLS 2005 forcefield to optimize all hydrogen bonding networks. Structure
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comparison between the cardiac and skeletal troponin was carried out by superpositioning the
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two structurally conserved Ca++ bound TnC. From this superpositioning, truncated hybrid and
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homotypic crystal structures were analyzed including: cTnC/cTnI, cTnC/sTnI, cTnC, cTnI, and
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sTnI. The cTnC:TnI regulatory complex was isolated by exclusion of all other residues in the
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crystal structure except: sTnI R115 – L140, cTnI R148 – L173, and each TnI was complexed
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with cTnC M1 – K90.
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Molecular Dynamics Simulation Molecular Dynamics (MD) simulations were performed for
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sTnI:cTnC; cTnI:cTnC; and sTnIH132A:cTnC using NAMD (78) version 2.6 with CHARMM27
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forcefield (63). Computational requirements were acquired using the University of Minnesota
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supercomputer BladeCenter Linux cluster. All the X-ray structures and derived mutants were
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prepared by CHARMM version c35b1. Each of the complex was solvated with explicit TIP3P
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water model (47) using a simulation solvent box with a 15 Å buffer region around the protein.
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Sodium counter ions were placed at 5 Å from the box boundary to neutralize the system. The
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solvated system was initialized with 5000 steps of conjugate gradient energy minimization with
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protein heavy atoms restrained at 50kcal/(mol•Å2). The system was then gradually heated with
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the same restraint from 25K to 300K at 25 K increment at 10 ps interval for 100 ps followed by a
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100 ps equilibration with gradual removal of the heavy atoms restraint at 10 ps interval under
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NVT condition. The final unrestrained equilibration was carried out for 100 ps followed by 40 ns
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of production simulation at 1 atm and 300K NPT condition. All simulations were carried with
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periodic boundary condition using Particle Mesh Ewald (PME) (27) with SHAKE (83) method
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employed to constrain bond lengths involving hydrogen atoms. The time step in the simulations
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was 2 fs with coordinates saved at 1-ps time intervals, resulting in a total of 40,000
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configurations for analysis. Two MD simulations were carried out for each of the modeled
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complexes by random initialization of the starting velocities.
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Interatomic distance calculations All distances are measured in angstrom over the 40 ns
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simulations for each of the simulated trajectories. For the wild type and H132A simulation of the
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sTnI:cTnC complex, the distance was evaluated between sTnI H/A132 (CA) and cTnC E19
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(OE2).
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Root mean squared fluctuations The root mean square fluctuations (RMSF) for the Cα atoms of
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each
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residue were calculated over each of the 40 ns simulated trajectories using a CHARMM script to
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evaluate the relationship
RMSF =
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~
1 T
( X i (t j ) − X i ) 2
∑
T t j =1
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where T is the trajectory time length, X i is the average position of each individual residue i at
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simulation time = j , and X i is the initial position of each residue.
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for TnC and TnI were evaluated independently of each other by superimposing all snapshots to
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the X-ray structure of each domain as the reference structure.
~
The residues fluctuations
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Theoretical isoelectric point calculations In the absence of crystal structures, values for the
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theoretical isoelectric point (pI) for a given amino acid sequence were calculated using the
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algorithm from ExPASy's Compute pI/Mw program(12).
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Theoretical pK calculations of Tn The general continuum electrostatics methodology widely
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used to calculate the energetics of proton transfer is described elsewhere(7, 8). Briefly, the
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difference between a sidechain’s pK and the pK of the corresponding model compound in free
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solution is determined by the combined effect of two distinct contributions to the total
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electrostatic (free) energy change. First is the “Born term” or desolvation penalty, which always
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penalizes burial of a charge inside a low dielectric medium. Second is the “background term”,
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which represents the electrostatic interactions of the group in question with all other fixed
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charges in the molecule not belonging to any titratable groups. These energy terms, as well as the
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matrix of site–site interactions Wij are estimated through a sequence of finite-difference Poisson-
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Boltzmann calculations in which sites in the protein and their corresponding model compounds
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have their charge distributions set to those of the protonated or deprotonated form, and suitable
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energy differences are taken. The methodology allows for a straightforward decomposition of the
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total computed pK value as a sum of contributions ΔpKi from all other groups in the protein,
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both titratable and non-titratable. Thus, the individual ΔpKi values can be used to characterize
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the strength of electrostatic interactions between neighboring groups in protein.
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Here we used a web-based implementation of the model available via H++ server(39)
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(http://biophysics.cs.vt.edu/H++/). In this work, the protein is treated as a low dielectric medium
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εin = 10, while the surrounding solvent is assigned a high dielectric constant εout = 80. Consistent
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with the premise of the continuum solvent models, all water molecules and ions present in the
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original PDB structures were removed before the calculation. The electrostatic screening effects
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of (monovalent) salt enter via the Debye-Huckel screening parameter κ which is set to
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correspond to physiological conditions of 0.145 M salt concentration. The choice of internal
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dielectric εin = 10 is justified for titratable groups that are not buried deeply inside the protein
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matrix, which is the case for the groups of interest in this study. Further justification for the
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above choice of εin was justified by comparison of pK values and protonation states of key
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groups computed by two completely different methodologies: H++ (continuum electrostatics)
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and PROPKA(58) (empirical). Specifically, the predictions of the large upward shift of HIS130
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pK differ by only about 0.2 pK units between the two methods.
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Computational alanine scanning: preparation of Ala mutant structures For each amino acid on
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the sTnI:cTnC binding interface we prepared its alanine substitution mutant by deleting all side-
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chain atoms beyond Cβ in the corresponding wild-type (WT) structure. Then, pK values of
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titratable groups in the WT and mutant structures were then calculated as described above, and
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their protonation states set accordingly assuming physiological pH (pH= 7.2). Computation of
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changes in binding free energies upon ALA substitutions A physical model for protein-protein
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binding free energies is used(49); the model is based on all-atom description and includes
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energetic contributions from VdW interactions, solvation and hydrogen bonding. Here we
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employ a web-based implementation (Robetta: http://robetta.bakerlab.org/alaninescan) of the
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model(50) with default parameter settings. The alanine scan was performed individually for each
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group on the binding interface of both partner proteins (sTnI:cTnC). The methodology computes
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change ΔΔG0 in protein-protein binding free energy upon alanine substitution of residue ”X” in
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position ”NN”: ΔΔG0ala = ΔGWT − ΔGMUT, where ΔGWT is the protein-protein binding free
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energy of the WT complex, and ΔGMUT is that of the corresponding XNNA mutant (e.g. H132A).
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Thus, ΔΔG0ala > 0 indicates destabilization of the complex in the mutant form relative to the WT.
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Most of the titratable groups in both binding partner proteins were previously determined
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to be in their “standard” protonation states; further, these states did not change upon protein-
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protein complex formation or ALA substitutions of individual interface groups. For these groups
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no additional calculations were necessary, the ΔΔG0 values computed by the Robetta server were
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reported directly in Supplemental Table 3.
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However, a calculation based on the H++ methodology reveals that the protonation state
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of HIS132 at pH=7.2 does change upon formation of the complex, resulting in the need to
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consider an additional pH-dependent contribution to ΔΔGala, which we denote as ΔΔGala(pH):
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ΔΔGala = ΔΔG0ala + ΔΔGala(pH). To compute this additional term, we follow earlier works(65,
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117) and express the total binding free energy as a sum of intrinsic and pH-dependent parts:
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ΔG = ΔG0 + ΔG(pH)
(1)
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where ΔG0 is the pH-independent (intrinsic) binding free energy computed when the site of
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interest is in a reference protonation state, and
⎡1 + 10 pK − pH ⎤
ΔG ( pH ) = − RT ln ⎢
⎥
pK F − pH
⎢⎣1 + 10
⎥⎦
C
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(2)
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where indices “C” and “F” denote, respectively, the complexed and free states of the two
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proteins respectively(65). We take the reference protonation state of HIS to be singly protonated,
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and GLU to be deprotonated, which are the “standard” protonation states assumed by Robetta
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model. With this assumption, ΔGWT and ΔGMUT computed by the model correspond to pH-
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independent, intrinsic ΔG0 in Eq. 1. Applying Eq. 2 to both WT and MUT, we obtain the
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following expression for the pH-dependent part:
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C
C
− pH
⎛ 1 + 10 pKWT
1 + 10 pK MUT − pH
⎜
ΔΔGala ( pH ) = − RT ln
×
F
− pH
⎜ 1 + 10 pKWTF − pH 1 + 10 pK MUT
⎝
⎞
⎟
⎟
⎠
(3)
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For HIS130 (and GLU19, for consistency), the values reported in Figure 4c are
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ΔΔGala = ΔΔG0ala+ΔΔGala(pH). For HIS132 the pH-dependent contribution is comparable to the
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intrinsic part ΔΔG0ala at the relevant pH=7.2; for GLU19 this additional contribution is very
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small since pK of this group is well below 7.2.
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Results and Discussion
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The principle goal of this study was to assess how cardiac Tn proteins have been
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structurally modified during evolution to accommodate the physiological requirements of
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mammalian myofilament function. To accomplish this, large scale phylogenetic analysis was
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performed by clustal W alignment of 104 primary amino acid sequences of troponin regulatory
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components including cTnT, cTnC, ssTnI, and cTnI from species throughout chordate evolution
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(Supplemental Figures 1-6, Supplemental Tables 1, 2). Table 1 lists species abbreviations
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summarizing the relevant taxonomy.
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Disease causing polymorphisms in the C-terminus of cTnI recapitulate non-mammalian
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physiology
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The positional relationship between disease-causing SNPs, functional domains and the
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secondary structural regions of TnI are outlined in Figure 1. Benign and disease causing single
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nucleotide polymorphisms (SNPs) have been identified along the full length of the human cTnI
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locus. Over 94% of known disease causing SNPs are located between the start of the IR domain
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and the end of the C-terminus (Figure 1b), also referred to as the regulatory domain of TnI(95).
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One of the physiological outcomes observed in these cardiomyopathies is increased myofilament
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calcium sensitivity(9, 20, 81, 101). The concentration of disease causing mutations within the
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regulatory region of TnI indicates that single amino acid alterations in this critical region of the
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molecule are not tolerated and have significant deleterious effects on TnI function that cause
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heart disease.
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Studies have suggested that, compared to the physiological constraints of mammals,
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environmental demands such as cold temperatures and hypoxic conditions require heightened
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myofilament calcium sensitivity to maintain sufficient cardiac performance in several fish (e.g.
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salmonids) and amphibian species(13, 32, 34, 35, 61, 62, 88, 89, 92). These observations indicate
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that heightened myofilament sensitivity to activating calcium is a feature of human cTn disease
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causing SNPs that recapitulates normal, healthy non-mammalian myofilament function.
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Phylogenetic analysis of slow skeletal and cardiac troponin I functional domains
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Human SNPs in TnI that cause pathologies recapitulating non-mammalian physiology
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provide a retrospective view of evolutionary selective pressures. Accordingly, these SNPs lend
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insights into the evolutionary basis for observed modifications in cardiac Tn during molecular
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evolution of the mammalian myofilaments(5, 48). Given the observed intolerance of SNPs in the
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C-terminus of TnI (Figure 1b), we hypothesized that evolutionary pressures tightly regulated
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changes in these regions to modify the performance of Tn in accordance with the evolution of
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mammalian requirements for sensitivity to activating calcium. To begin, the primary amino acid
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sequence of subdomains in TnI were analyzed to understand the phylogenetic basis for
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differences within these functional regions of the molecule between chordate species (Figure
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2a,b).
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Isoforms of TnI are differentially expressed among chordate species. In amphibians the
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cardiac TnI isoform has been found to be the only TnI gene expressed at the onset of heart
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formation in the larva and continuing in the adult heart of anuran species(25, 104, 105).
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However, in avian and mammalian species the slow skeletal TnI gene is transiently expressed
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during heart development and is replaced in the adult heart by the cardiac TnI gene(77, 84, 91).
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Based on these different expression profiles, we hypothesized that comparative analysis of TnI
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isoforms expressed in the heart would provide a basis with which to interpret modifications in
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TnI during evolution of chordate cardiovascular systems.
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To discern evolutionary pattern differences among the functional domains of TnI,
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discrete segments of the molecule were aligned and compared across all species. The domains of
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TnI analyzed included the N-terminus (1-44), N-terminal TnC binding domain (45-65), IT arm
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(91-136), inhibitory region (IR) (138-149), switch arm (152-172) and the C-terminus (153-210).
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To determine if amino acid sequences within these functional domains were different between
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ssTnI and cTnI across chordate organisms, sequence alignment analysis was performed on
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species where both isoforms are known (Figure 2a). This analysis showed that, in contrast to N-
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terminal domains, regions within TnIreg (residues 138-210) all showed a significant reduction in
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sequence conservation within the cTnI isoform compared to ssTnI (P < 0.05). Furthermore, the
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cTnI switch arm had the greatest difference in sequence percent identity compared to the ssTnI
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switch arm (Δ percent ID = -20.1 ± 1.9%, P < 0.05 compared to all other domains).
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To understand how this change in switch arm conservation correlates with specific
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phylogenic groupings, the ssTnI/cTnI sequence percent identity was compared in a pairwise
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fashion within each class (mammalian, avian, amphibian) (Figure 2b). This analysis showed that
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among amphibians the switch arm of ssTnI and cTnI isoforms are more similar than is the case
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for birds. The greatest divergence (lowest percent ID) in ssTnI:cTnI sequence conservation was
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observed in mammals. In contrast, other functional regions such as the N-terminal TnC binding
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domain showed no difference in ssTnI:cTnI sequence conservation among the taxa studied
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(Figure 2b).
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These phylogenetic data indicate that the switch arm was a site of rapid ssTnI:cTnI
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sequence divergence exclusively within the mammalian lineage. A vast literature supports the
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conclusion that the properties of the TnI switch arm can markedly alter the mechanism by which
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troponin regulates the contractile properties of the myofilament (22, 38, 60, 69, 71, 95, 103, 107,
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109-113, 115).
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Analysis of the amino acid composition of the TnI switch arm
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We next sought to determine how the mammalian switch peptide was modified. To
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begin, the theoretical isoelectric point (pI) was calculated for the switch arm fragment for each
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class (Figure 3a,i). These data show that the ssTnI switch arm pI did not change across any
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classes. In contrast, the pI for the cTnI switch arm decreased from amphibian (9.5 ± 0.5) and bird
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(8.75 ± 0.0) species to mammals (6.76 ± 0.0, P < 0.05 vs. mammalian ssTnI). These data also
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show no significant difference between the TnI isoform (ssTnI vs. cTnI) pI for amphibian and
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avian species. Comparatively, there is a significant reduction in the pI for mammalian ssTnI vs.
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cTnI (9.99 ± 0.0 vs. 6.76 ± 0.0, P < 0.05). These data are supported by crystal structure pI
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analysis (Supplemental Table 3). Linear regression analysis showed that evolutionary pressure to
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modify the switch arm peptide sequence between ssTnI and cTnI are significantly related to
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alterations in the pI of the domain (r2 = 0.84, P = 0.0002) (Figure 3a, ii). Evidence that the ssTnI
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switch arm sequence composition and pI are not different across classes indicates that this
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isoform is not being modified during chordate evolution. In contrast, modifications in ionizable
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residues in the cTnI switch arm are altered during evolutionary peptide adjustments of the
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mammalian cTn complex.
374
375
Sequence analysis of the troponin I switch arm
376
377
To further study the observed modification of the switch arm in cardiac TnI from
378
amphibians to avian and mammalian species, predicted protein and mRNA sequences of cTnI
379
from numerous chordate species were collected. In addition, putative TnI sequences were
380
identified by BLAST analysis of genomes from Anolis carolinensis (Lizard), Taeniopygia
381
guttata (Zebra Finch), Cavia porcellus (Guinea Pig), and Callithrix jacchus (Marmoset) (see
382
Supplemental Table 2 and supplemental Figures 2 and 3). Isoform-dependent variation in
383
ionizable residues between ssTnI and cTnI in the switch arm were studied (Figure 3b). This
384
analysis showed that a nonpolar hydrophobic moiety at position 164 in the switch arm of cTnI is
385
100% conserved in mammals but was found to be a histidine in all other chordate species and
386
TnI isoforms (with the exception of zebra finch cTnI). Although alanine 164 was identified in all
387
other mammals, a proline residue was surprisingly identified in the predicted platypus sequence.
388
Phylogenetically, platypus, an egg laying mammal, is situated early in the mammalian lineage
389
(Supplemental Figure 1). This modification of an amino acid that is perfectly conserved through
390
chordates prior to the bird/mammal divergence and subsequently altered (charged to uncharged
391
or vice versa) only in mammals and thereafter perfectly conserved, was not observed at any other
392
locus in the entire troponin complex (cTnI, ssTnI, cTnT, or cTnC). Such a significant level of
393
residue conservation suggests that the transition from a hydrophilic and polar amino acid to a
394
hydrophobic and nonpolar moiety within the switch arm is likely a key modification in Tn during
395
evolution of the mammalian heart. This is supported by studies showing the importance of this
396
TnI histidine button in myofilament function(22, 23, 75, 76, 112). Analysis of selective pressure
397
at the nucleotide level indicates that the mammalian cTnI switch arm is under significant
398
purifying selection suggesting that the current peptide composition has long been highly
399
favorable for the proper regulatory functions of troponin in the mammalian heart (Supplemental
400
Table 4).
401
402
Crystal structure analysis of the calcium saturated troponin complex
403
404
The phylogenetic data indicate that the switch arm of amphibian cTnI is very similar to
405
ssTnI from across all classes in terms of amino acid sequence and isoelectric point (Figure 3a, b).
406
Studies have also shown that cTnI physiology seen in fish and amphibians is functionally
407
recapitulated by ssTnI in the mammalian heart in vivo and in vitro(1, 33, 57, 67, 68, 89, 102, 113,
408
114). Importantly, what we know about mammalian ssTnI may therefore assist in our
409
understanding of the structure/function relationship of anuran and fish cTnI.
410
In recent years the crystal structures of the calcium saturated cardiac Tn complex(95)
411
(PDB: 1J1E, 3.3 Å) and fast skeletal Tn complex(103) (PDB: 1YTZ, 3.0 Å) have been reported.
412
We assume that the regulatory domain of the putative ssTn complex mirrors that of the reported
413
fsTn complex (for justification of this assumption and structural orientation see Supplemental
414
Figure 7). We propose that comparative molecular modeling of sTn and cTn through hybrid
415
(sTnI(103) with cTnC(95)) and homotypic (cTnI with cTnC(95)) atomic resolution crystal
416
structures provides insight into evolutionary modifications that occurred in cTn to accommodate
417
the functional requirements of the mammalian heart.
418
We find the structural alignment of the sTnI and cTnI crystal structures in complex with
419
cTnC accentuates the differences in the regulatory interaction between these protein complexes
420
(Figure 4a). Comparison of the sTn and cTn crystal structure shows a marked alteration in the
421
position of TnI helix 4 between sTnI and cTnI relative to helix A and the hydrophobic patch of
422
TnC (Figure 4a). The structural alignment indicates that H3 has similar positioning in the two
423
complexes. The structure of skeletal TnI indicates that unstructured helix 4 has His132 engaging
424
helix A of TnC in very close proximity to a key acidic residue, Glu19, in the hydrophobic patch
425
of TnC. This analysis shows that TnI H132 (protonated, pKa = 8.25) and TnC E19 (pKa = 2.51)
426
are close enough to develop a salt bridge (Supplemental Table 3, Figure 4a, inset). This
427
computational analysis indicates that the theoretical binding energy of this interaction is 5.24
428
kcal/mol, the strongest interatomic bonding observed between any two ionizable groups analyzed
429
in these crystal structures (Figure 4b). Decomposition of pK data indicates that the pH titration
430
characteristics of H132 and E19 are significantly influenced by the reciprocal electrostatic
431
interactions between these residues (Supplemental Figure 8a,b and Supplemental Table 3). The
432
characteristics of this key interaction between cTnC and sTnI (and thus its functional
433
importance) are also observed in another unique histidine-aspartate interaction (H146β-D94β) in
434
hemoglobin that forms a salt bridge and singularly contributes to about half of the maximal
435
alkaline Bohr effect of human HbA(10).
436
In contrast to the sTn structure, at the H3-H4 linker and into the structured H4 of cTnI
437
Ala164 translocates away from helix A and the hydrophobic patch of TnC (Figure 4a). Residue
438
ionization titration analysis confirmed the hypothesis that cTnC and cTnI have limited
439
electrostatic contact at this critical interaction site (Figure 4b, Supplemental Table 3,
440
Supplemental Figure 8). Crystal structure analysis showed the strongest electrostatic contacts
441
within the cTnI:cTnC complex to be over four times lower than E19-H132 of cTnC-sTnI.
442
To gain molecular insights into the marked differences between the sTnI-cTnC and cTnI-
443
cTnC regulatory complexes, computational simulations were carried out in nanosecond timescale
444
to study the molecular dynamics and stability of the two complexes in atomistic detail. These
445
data show that the switch arm (unstructured H4 in particular) retains strong interface contacts
446
between sTnI and cTnC but becomes highly mobile, molecularly untethered, in the cTnI-cTnC
447
structure (Supplemental movies 1 (sTnI:cTnC) and 2 (cTnI:cTnC)).
448
The performance characteristics of the troponin complex have been shown to be
449
markedly different between myofilaments containing sTnI vs. cTnI(59, 108, 110, 113). 2D NMR
450
spectroscopy analysis of amide chemical shifts during calcium titration has shown that the switch
451
peptide dissociation constant for cardiac TnI is six times weaker than that of skeletal TnI(59).
452
Transgenic and gene transfer studies have shown that myofilaments containing ssTnI have
453
sarcomere dynamics including hypercontractility, slow relaxation, and increased propensity for
454
arrhythmias compared to myofilaments containing cTnI(9, 112, 118). Furthermore, consistent
455
with the concept of a pH-responsive histidine button, TnI proteins with a histidine moiety in the
456
switch arm (anuran cTnI, mammalian ssTnI or mammalian cTnI A164H) perform as a molecular
457
rheostat capable of maintaining inotropy in the context of severe acidosis or other pathologies(3,
458
18, 22, 75, 76, 102, 112-114). Myofilaments containing cTnI with an alanine in the switch arm
459
(A164), by contrast, are highly susceptible to acidosis induced contractile failure. Taken
460
together, these structural and functional data indicate that significant alterations in the peptide
461
composition and, consequently, tertiary protein structure of the cardiac Tn regulatory interaction
462
markedly alter the functional properties of Tn.
463
464
We next tested the hypothesis that breaking of the His132-E19 interaction would
markedly decrease the thermodynamic affinity between sTnI and cTnC. To test this,
465
computational alanine scanning was performed to calculate the change in the binding free energy
466
(ΔΔG) caused by alanine substitution of all residues involved in the intermolecular interface
467
contacts of the sTnI-cTnC structure (Figure 4c). In cTnC, alanine substitution of E19 was found
468
to contribute most to the ΔΔG (ΔΔG E19A = 0.91kcal/mol). However, the greatest impact on the
469
binding free energy among all residues engaged at the cTnC:sTnI interface was observed with
470
the sTnI H132A substitution (ΔΔG H132 = 2.46 kcal/mol). This thermodynamic analysis
471
supports the conclusion that alanine substitution of sTnI H132 was the most effective mechanism
472
of reducing the binding free energy at the interface of the calcium saturated sTnI-cTnC structure.
473
Interatomic distances were calculated between the cTnC E19 (OE2) and residue 132 of
474
either WT sTnI (ND1) or sTnI H132A (CB) across a 40 nanosecond atomic resolution molecular
475
dynamics simulation (Figure 5a,b and Supplemental video 3 (sTnI H132A:cTnC)). These data
476
show the WT sTnI:cTnC structure retained a highly stable interface mediated by the high energy
477
intermolecular salt bridge interaction between His132:E19. In contrast, there was a marked
478
increase in the distance between cTnC E19 and sTnI residue 132 in the alanine substituted
479
structure indicating a significant structural interface separation and destabilization of the
480
complex (Figure 5b). Positional root mean square fluctuations (RMSF) of all residues during the
481
40 ns MD simulation also indicate structural untethering within the sTnI H132A switch arm
482
compared to WT sTnI (Figure 5c). These MD simulations were replicated with a different
483
randomly assigned starting velocity and the results confirmed our initial observation
484
(Supplemental Movie 4). Functional data support these in silico studies. A recent report by
485
Westfall et al has shown that ssTnI H132A significantly reduces the calcium sensitivity of the
486
myofilaments (pCa50) and enhances the relaxation performance of myocytes similar to the
487
functional properties observed with myocytes expressing mammalian cTnI(112).
488
Given our initial assumption that sTnI recapitulates ectothermic cTnI, the molecular
489
modeling data indicate that there was evolutionary pressure in the mammalian taxa to favor cTnI
490
A164 in order to reduce the binding free energy (ΔΔG) at the interface of cTnI and cTnC. This
491
was accomplished by eliminating the key high energy electrostatic interaction between TnI and
492
cTnC (H132-E19) making H4 and the C-terminal domain of TnI molecularly untethered.
493
Together with published functional data(112), the phylogenetic and molecular modeling data
494
here indicate that the modification in the switch arm of cTnI between amphibians (histidine) and
495
mammals (alanine) significantly reduced myofilament calcium sensitivity through enhanced
496
relaxation of cTn in the mammalian cardiovascular system. The proline residue observed in
497
platypus cTnI accentuates this point. Proline is the archetypal helix breaker and is also nonpolar.
498
Thus we posit that structurally and functionally, the platypus proline at codon 164 is an excellent
499
intermediate to enhance myofilament relaxation of cTn through disruption of the histidine-
500
glutamate salt bridge predicted to occur in fish and anuran species.
501
The hypothesis that this histidine vs. alanine residue in the switch arm of cTnI is
502
important in the evolutionary divergence of ectotherm vs. mammalian physiology could be tested
503
by an alanine substituted cTnI into fish myocytes. If our hypothesis is correct then an alanine
504
residue in cTnI of fish (e.g. zebrafish) would markedly diminish calcium sensitivity of the
505
myofilaments in vitro and would likely not be sufficient to support normal physiological
506
requirements of that organism in vivo. Alternatively, if this model is correct, expression of
507
ectothermic cTnI in mammalian myocytes would increase calcium sensitivity of the
508
myofilaments.
509
510
Increased myofilament calcium sensitivity in non-mammalian chordates
511
512
Previous reports have shown that residues at the N-terminus of cTnC are required for
513
differentially modulating the calcium binding affinity of TnC between endotherms and some
514
ectotherms (Supplemental Figure 6)(32, 33, 35). It has been suggested that, in at least one
515
ectotherm species, trout, four residues (GrP sequence: N2, I28, Q29, D30) are responsible for
516
decreasing the energy barrier required for conformational changes in the N-terminus of TnC
517
which increased calcium binding affinity at EF hand site II(32, 33, 35). In endotherms (birds and
518
mammals), these residues have been converted with high conservation to D2, V28, L29, and G30
519
and markedly reduce the calcium binding affinity of cTnC(33). In mammals, failure to modify
520
these residues results in cardiomyopathy. Studies have shown that a single amino acid
521
substitution L29Q in human cTnC results in hypertrophic cardiomyopathy the underlying cause
522
of which may be a significant increase in myofilament calcium sensitivity, although an
523
alternative possible explanation involving potential alterations in the PKA-dependent phosphor-
524
cTnI:cTnC engagement has also been suggested(6, 44, 61, 85). Other pathogenic peptide
525
deviations in mammals associated with increased myofilament sensitivity to activating calcium
526
have been found in SNPs within other regions of cTnC as well as cTnT(9, 28, 55, 72, 73, 79, 81,
527
97, 98, 116). Taken together, these studies indicate that evolutionary selective pressure to modify
528
cTn occurred within the adult mammalian heart to reduce myofilament calcium sensitivity to an
529
extent markedly below ectothermic chordates.
530
531
532
Enhanced relaxation of cardiac troponin required for mammalian heart function
533
There is a significant body of literature providing comparative analysis of cardiovascular
534
function between chordate species (11, 17, 29, 42, 43, 53, 88, 89, 94, 104 , 105). These studies
535
reveal important details about the differences in physiological requirements that necessitate
536
unique mechanisms of cardiovascular support among chordate species. The current study
537
contributes to this knowledge base by providing the first integrated model for molecular
538
evolution of the cardiac troponin complex. These data show that cTn modifications enhanced the
539
relaxation performance of the thin filament regulatory complex and may have evolved to
540
accommodate unique demands for myofilament function of the adult mammalian heart (Figure
541
6).
542
Compared to adult mammalian myofilaments, the expression of TnI isoforms with
543
inherently high calcium sensitivity in the developing hearts of a range of chordate species
544
(amphibian cTnI and bird/mammalian ssTnI) (25, 68, 84, 92, 113) indicates that heightened
545
calcium sensitivity and pH resistivity of the myofilaments may be important for heart
546
development in chordate species (Figure 6). Several studies support this assertion. First, the fetal
547
heart has high lactate levels and relies on glucose metabolism(64). Second, there is evidence that
548
the critical cardiac developmental transcription factor Nkx2.5 is regulated by hypoxia-inducible
549
factor (HIF-1α)(74). Third, immature sarcoplasmic reticulum and calcium cycling in the
550
developing heart require heightened sensitization of the myofilaments to establish sufficient
551
nascent heart pump function(31, 86).
552
However, in the adult heart, requirements for proper regulation of cardiac output are
553
markedly different between chordate species. Numerous factors intrinsic and extrinsic to the
554
heart affect the efficiency of heart rate and stroke volume to sustain cardiac output.
555
Cardiomyopathic SNPs identified in all members of the human heterotrimeric cardiac troponin
556
complex have been found to cause pathologies characterized by a marked increase in
557
myofilament calcium sensitivity(19, 20, 26, 44, 46, 61, 87, 106). Together, these studies show a
558
strong relationship between cardiomyopathic SNPs in troponin and heart failure phenotypes
559
including diastolic dysfunction, arrhythmias, and sudden cardiac death.
560
In the context of chordate evolution, our current understanding of fish and amphibian
561
physiology support the need for heightened myofilament calcium sensitivity with pH-resistivity
562
compared to mammals(62, 88-90, 92). Similar to cTnI, other proteins have shown analogous
563
adaptions in these species. For example, hemoglobin in many fish has an exacerbated Bohr effect
564
known as the Root effect, an extreme form of pH sensitive Hb O2 binding(10, 14, 82). On the
565
part of regulating cardiac contractility during hypoxia, modifications in intracellular pH in
566
response to temperature shifts, termed α-stat regulation, is thought to maintain cardiac
567
myofilament function in ectothermic and poikilothermic species by keeping the fractional
568
dissociation of histidine imidazole groups constant(16, 36). The N-terminal NIQD motif of cTnC
569
and the switch arm peptides of cTnI seen in fish and amphibians may also provide a substrate to
570
support the required cardiovascular demands of ectotherms (Figure 6).
571
The observation that pathogenic peptide deviations (either naturally occurring SNPs(20,
572
61) or in situ protein mutagenesis (32, 112)) cause cell physiological effects including
573
hypersensitive myofilaments indicates that, compared to other non-mammalian chordate
574
phylogenies, reduced myofilament sensitivity to activating calcium was a necessary adaptation
575
within the mammalian heart. Phylogenetic alignment data show the emergence of protein kinase
576
(PKA and PKC) phosphorylation sites at the N-terminus of TnI (S23, 24) at the level of
577
amphibians (Supplemental Figure 2). One of the major purposes for protein kinase
578
phosphorylation of these residues is to reduce the calcium sensitivity of the myofilaments in
579
coordination with changes in heart rate(56, 70, 96, 118). Although covalent modifications of TnI
580
emerged prior to mammals, this mechanism for modulating myofilament function was evidently
581
insufficient for mammalian cardiovascular function. As a consequence, this study supports the
582
hypothesis that in the adult mammalian heart coordinated residue modifications affecting the key
583
regulatory interaction between cTnC and cTnI enhanced the intrinsic relaxation potential of
584
troponin independent of covalent modifications.
585
Structure and function data indicate that, in endotherms, these coordinated residue
586
modifications occurred in cTnC by markedly decreasing the calcium binding affinity at EF hand
587
site II(32, 33, 35). However, among all mammalian sequences identified, this study provides new
588
evidence of a critical hydrophobic and non-polar (primarily alanine) moiety substituted for
589
histidine in the switch arm disrupting a strong intermolecular electrostatic interaction at the
590
interface between cTnI and cTnC in the calcium saturated state (Figure 6). In silico molecular
591
dynamics alanine scanning simulations shown here together with recent functional data(112)
592
support a model that, only within mammals, this His to Ala substitution reduced myofilament
593
calcium sensitivity by making cTnI molecularly untethered from cTnC thus reducing the
594
thermodynamic potential at the intermolecular interface (ΔΔG). These data also indicate that loss
595
of a therapeutically effective pH responsive histidine button in cTnI(22, 75, 76) was necessary to
596
reduce myofilament sensitivity to activating calcium(112). As such, this study makes use of
597
disease causing mutations in troponin as they provide insight into the evolutionary physiological
598
adaptations of the mammalian cardiac myofilament. The acquisition of an alanine moiety in the
599
switch arm of troponin I with coordinate residue modifications in TnC reveal the need for
600
reduced cardiac myofilament calcium sensitivity in mammalian species.
601
602
603
604
Acknowledgements
605
606
We thank the labs of the University of Minnesota Lillehei Heart Institute for questions
607
oriented toward the phylogenetics of cardiac troponin that stimulated this study. We thank
608
Nobubelo Ngandu, Dr. Cathal Seoighe, and Dr. Simon Frost for assistance with interpretation of
609
selective pressures on the cTnI molecule. We thank Dr. Robert Kulathinal for his expertise in
610
compensatory pathologic mutations in evolution. We also thank the University of Minnesota
611
Supercomputing Institute for providing the computational resources. This work was supported by
612
the National Institutes of Health (RO1 HL 059301 to J.M. and R01 GM076121 to A.O.), the
613
American Heart Association (0715632Z to N.P.), and the University of Minnesota Center for
614
Drug Design (YS). Current address for NJP: University of Washington, Center for
615
Cardiovascular Disease and Regenerative Medicine, 815 Mercer, Seattle, WA 98117.
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954
Table 1. Species taxonomy and abbreviations
955
Mammals
Human
Homo sapiens
H
Sumatran orangutan Pongo abelii
SO
Chimpanzee
Pan troglodytes
Ch
Rhesus
Macaca mulatta
Rh
Marmoset
Callithrix jacchus
Ma
Bovine
Bos taurus
B
Horse
Equus caballus
Ho
Goat
Capra hircus
G
Sheep
Ovis aries
Sh
Pig
Sus scrofa
P
Dog
Canis lupus familiaris
D
Cat
Felis catus
C
Guinea Pig
Cavia porcellus
GP
Rabbit
Oryctolagus cuniculus
Rab
Rat
Rattus norvegicus
R
Mouse
Mus musculus
M
Opossum
Monodelphis domestica
O
Platypus
Ornithorhynchus anatinus
Pl
Reptile
Lizard
Anolis carolinensis
L
Birds
Chicken
Gallus gallus
Ch
Quail
Coturnix coturnix
Q
Amphibians
Turkey
Meleagris gallopavo
T
Zebra Finch
Taeniopygia guttata
ZF
Xenopus laevis
Xenopus laevis
Xl
Xenopus tropicalis
Xenopus (silurana)
Xt
tropicalis
Fish
Bullfrog
Rana catesbeiana
BF
Marine Toad
Bufo marinus
MT
Salmon
Salmo salar
S
Zebrafish
Danio rerio
Z
Green pufferfish
Tetraodon fluviatilis
GrP
Dinosaur eel
Polypterus senegalus
DE
Arctic lamprey
Lethenteron japonicum
AL
Halocynthia roretzi
As1
Ascidian 2
Ciona intestinalis
As2
Amphoxius
Branchiostoma belcheri
A
Invertebrates Ascidian 1
956
957
958
959
960
Figures
961
962
Figure 1. Structure and function analysis of cardiac troponin I. Schematic diagram of the
963
heterotrimeric troponin complex including troponin I (TnI), troponin C (TnC), and troponin T
964
(TnT). Coloring of structural components in TnI (e.g. helix 1- 4) in (a) are coordinated with
965
functional domains including the N-terminus, the N-terminal TnC binding domain, the IT arm,
966
the inhibitory region (IR), the switch arm, and the C-terminus as shown in (b). (b) Specific
967
amino acid loci of the human cardiac troponin I gene sequence with known disease causing
968
single nucleotide polymorphisms (SNPs); hypertrophic cardiomyopathy (green), dilated
969
cardiomyopathy, recessive (blue), dilated cardiomyopathy, autosomal dominant (brown), and
970
restrictive cardiomyopathy (red). Stacked bars at a single locus indicate multiple known
971
mutations at that site. 1 variant of uncertain effect and 6 polymorphisms between residues 39-
972
136 are not shown. SNP data were acquired from the Harvard cardiogenomics website
973
(genetics.med.harvard.edu) and recent reports(15).
974
975
Figure 2. Phylogenetic analysis of TnI functional domains. (a) Direct pairwise comparison by
976
Clustal W of sequences within specific functional domains of slow skeletal and cardiac TnI only
977
from species for whom both isoforms have been sequenced; these include H, P, Rab, R, M, Ch,
978
Q, Xl, Xt, BF, and S. * P < 0.05 by students t-test comparing ssTnI to cTnI within a given
979
functional subdomain. (b) Comparison of sequence percent identity between ssTnI and cTnI for a
980
given species and collated based on class including mammal (H, P, Rab, R, M), bird (Ch, Q), or
981
amphibian (Xl, Xt, BF). Differences between mammal, bird, and amphibian TnI isoforms are
982
shown for both the switch arm and N-terminal TnC binding domain. P < 0.05 based on 1 way
983
ANOVA within each subdomain.
984
985
Figure 3. Theoretical isoelectric point (pI) analysis and sequence analysis of the troponin I
986
switch arm. (a) Analysis of theoretical pI alterations in the switch arm between ssTnI and cTnI
987
for species where both sequences are available (H, P, Rab, R, M, Ch, Q, Xl, Xt, and BF). (a, i)
988
Analysis of the average pI of the ssTnI and cTnI switch arm for amphibian, avian, and
989
mammalian species. * P < 0.05 vs. all other groups by 1 way ANOVA. (a, ii) Linear regression
990
analysis showing the relationship of the net difference in pI and sequence percent identity
991
between ssTnI and cTnI within the switch arm. Precise overlap of values within classes reduced
992
the number of visible points to 5 in comparison to the 10 calculations made (1 for each species).
993
(b) Alignment of troponin I protein sequences expressed in numerous chordate species. Analysis
994
was performed by the Clustal W method. Sequences are organized by isoform and within each
995
isoform by species based on taxonomic classification. The key histidine to alanine amino acid
996
modification is also shown (yellow). The platypus proline is highlighted as the potential
997
evolutionary intermediate residue between histidine in fish and anuran species and alanine in all
998
other mammals. For alignment analysis of the full TnI molecule see Supplemental Figure 2. The
999
finding of a histidine moiety at position 164 in zebrafish cTnI HC by Fu et al (30) was recently
1000
reported and, consequently, not included in this analysis. The finding supports the model
1001
outlined by this report and the sequence alignment shown in Supplemental Figure 9.
1002
1003
1004
Figure 4. Molecular modeling and structural analysis. (a) Atomic level alignment of the crystal
1005
structures for sTnI and cTnI in complex with the cTnC focusing on regulatory interaction site
1006
between the switch arm of TnI and the N-terminal lobe of cTnC. cTnC (red), sTnI (blue), cTnI
1007
(yellow). Inset image highlights the critical interaction between sTnI H132 and cTnC E19. (b)
1008
Intermolecular interaction between ionizable groups (kcal/mol) in TnI and TnC are shown for the
1009
sTnI-cTnC structure (top) and cTnI-cTnC structure (bottom). Interactions are organized into
1010
intramolecular interactions within TnI (left) and cTnC (middle) and intermolecular interactions
1011
at the interface between TnI and cTnC (right). The strength of interaction cTnC E19 – sTnI H132
1012
is indicated in orange. Vertical bar = 1 kcal/mol. (c) Alanine scanning analysis of the change in
1013
binding free energy (ΔΔG) among all residues at the sTnI:cTnC regulatory interface. Important
1014
contributions to ΔΔG by cTnC E19 and sTnI H132 are shown in orange.
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1016
Figure 5. Molecular dynamics simulation modeling of interface separation caused by alanine
1017
substituted sTnI. (a) Structural alignment of the initial X-ray crystal structure t = 0 ns; WT sTnI
1018
structure t = 40.789 ns; and sTnI H132A structure t = 40.789 ns showing interface separation
1019
only in the alanine substituted structure . (b) Instantaneous (left) and calculated mean (right) of
1020
the interatomic distance between cTnC E19 (OE2) and residue 132 of WT sTnI (H132 ND1,
1021
blue) or sTnI H132A (CB, red). Mean data are derived from average atomic distance
1022
measurements acquired during two independent simulations. (c) Atomic level positional root
1023
mean square fluctuations (RMSF) of each residue during the 40 ns MD simulation showing a
1024
marked increase from WT only within the switch arm of sTnI H132A. * P < 0.05.
1025
1026
Figure 6. Proposed model for molecular evolution of cardiac troponin during chordate evolution.
1027
Progressive modifications in organismal cardiovascular requirements resulted in marked changes
1028
in heart structure and functional regulation to meet evolving whole animal physiologic demands.
1029
Troponin isoforms expressed in species identified early in chordate evolution (e.g. fish (cTnI)
1030
and amphibians(cTnI)) together with saurian species and developmental isoforms from all
1031
classes (fish (cTnI), amphibian (cTnI), avian (ssTnI), and mammalian (ssTnI)) are hypothesized
1032
to have a high energy histidine-glutamate interaction in the regulatory intermolecular interface
1033
between cTnI and cTnC. This results in myofilaments with heightened calcium sensitivity and
1034
reduced pH sensitivity. During evolution of the mammalian adult cardiovascular system
1035
(developmental isoform transition to cTnI), the cardiac troponin I switch arm evolved to contain
1036
a proline moiety in place of the histidine (as seen with the platypus) that fundamentally altered
1037
the critical regulatory interaction between cTnI and cTnC. Together with all other mammals
1038
(which have A164), this resulted in myofilaments with reduced calcium sensitivity and increased
1039
pH sensitivity compared to non-mammalian chordates.
1040
1041
1042
1043