Supplementary Information
Hamès et al.
Structural basis for LEAFY floral switch function and similarity with
helix-turn-helix proteins
Supplementary Information
This file includes:
Supplementary Figures 1 to 5
Supplementary Table 1
Supplementary Methods
S1
Supplementary Information
Hamès et al.
Supplementary Figures
Supplementary Figure 1: Comparison of the initial experimental SIRAS electron
density map after solvent flattening (A) and the final 2Fo-Fc electron density map (B).
Both maps are contoured at 1.9 .
S2
Supplementary Information
Hamès et al.
Supplementary Figure 2:
EMSA with wild-type and mutant AP1 probes (10 nM) and wild-type LFY-C
(concentrations in nM). Mutations in binding sites resulted in a reduction of both
binding affinity and cooperativity as shown by the increased monomeric complex (m)
concentration and reduction of the dimeric one (d). 5’ to 3’ sequence of the DNA upper
strand is indicated on the right of the EMSA. The dots indicate the center of the pseudopalindromic binding site.
Supplementary Figure 3: EMSA with wild-type and mutant LFY-C proteins
(concentrations in nM). AP1 DNA concentration is 10nM. Positions of dimeric (d) and
monomeric (m) complexes are indicated. These mutations had been found to abolish
DNA binding in previous experiments, probably because the binding was only tested
under conditions where the wild-type protein shifts only small amounts of the DNA
probe
S3
Supplementary Information
Hamès et al.
Supplementary Figure 4: Stereo diagram of the interactions between R237 and base
pairs 7 and 8 observed in the AP1 (top) and AG-I (bottom) sites. For the AP1 site
depicted is the experimental SIRAS electron density map after solvent flattening
together with the final model. For the AG-I site no experimental phases are available.
S4
Supplementary Information
Hamès et al.
Instead, depicted is a simulated annealing omit map and the final model calculated with
the first three LFY residues (R237 to H239) and base pair 7 lacking from the model.
Both electron density maps are contoured at 1.1 . The side chain of Arg237 is in
contact distance to base pairs deviating from 2-fold symmetry (bp+9 and +7 in AP1;
bp+ 7 in AG-I). In the AP1 site Arg237 NH1 and NH2 interact with the exocyclic O2 of
Thy+8 in the AP1 site. In one halfsite Arg237 NH1 contacts the O2 of Cyt+9 of the
same strand, while in the other halfsite Arg237 NH2 contacts O2 of Cyt-7 of the
opposite strand (depicted here). Arg237 possesses well-defined side chain density
indicating a unique conformation despite the differences in the two halfsites. In the AGI site, Arg237 NH1 contacts the O2 of Thy+8, but is too far away to form direct
hydrogen bonds with the bases in base pair +7. In the AG-I site the electron density for
the Arg237 side chain is slightly less well defined as in the AP1 site, but we do not find
evidences for alternative conformations of this side chain.
S5
Supplementary Information
Hamès et al.
Supplementary Figure 5: Quantitative analysis of cooperativity
Quantifications of free DNA (blue), monomeric complex M (red) and dimeric complex
D (green) were obtained from experimental data shown in Figure 5 and plotted as
circles, squares and diamonds, respectively. Calculated fits are represented as plain lines
following the same color code.
S6
Supplementary Information
Hamès et al.
Supplementary Table 1: Sequences of oligonucleotides used for EMSA. Only forward
sequences are indicated (5’ to 3’). Mutant bases are underlined. The guanine
corresponding to the center of the pseudo-palindromic binding site is depicted in bold.
Oligonucleotide
Sequence
AP1 WT
TTGGGGAAGGACCAGTGGTCCGTACAATGT
AP1 m1
TTGGGGAAGGAAAAGTGGTCCGTACAATGT
AP1 m2
TTGGGGAAGGACCAGTAATCCGTACAATGT
AP1 m3
TTGGGGAAGGAAAAGTAATCCGTACAATGT
AP1 m5
TTGGGGCAGGACCAGTGGTCCGGACAATGT
Supplementary Methods
Determination of dissociation constants.
Signals were quantified from the gels shown in Figure 5 using ImageQuant software
(Molecular dynamics, Sunnyvale, CA). The sum of the signals per lane was normalized
to 100% and the concentrations of free DNA (A), monomeric (M) and dimeric (D)
complexes were calculated as a fraction of total fluorescent DNA concentration (50 nM
in these experiments). The fits were performed assuming Kd1 is the same for all 4
proteins (LFY-C, H387A, R390A and H387A R390A). Without this assumption, Kd1
values were found to be very similar to each other but with larger confidence intervals.
Model variables and parameters.
Variable
Symbol
Unit
Total DNA
A0
nM
Total protein
C0
nM
Free DNA
A
nM
S7
Supplementary Information
Hamès et al.
Free protein
C
nM
Protein.DNA
M
nM
(Protein)2.DNA
D
nM
First dissociation constant
Kd1
nM
Kd2
nM
Second
dissociation
constant
Chemical reaction
Mass-action constraints
K d1 
A C M

C M  D

K d2 

AC
M
MC
D

Conservation relation
Total DNA
A0  A  M  D
Total protein
C 0  C  M  2  D

 solution.
Mathematical analysis and least-square
From the mass-action conditions, we get the concentration of the two complexes M and
D as a function of free DNA and protein concentrations:
S8
Supplementary Information
Hamès et al.
M 

D 
AC
Kd1
MC  AC
2
Kd2  Kd1
Kd2
The DNA conservation condition together with the two above relations, gives free DNA

as function of free protein.
A0
A 
C  C

2
1+
Kd1
Kd2  Kd1  A0
Kd2  Kd1 +Kd2  C  C
2
Kd2  Kd1
(1)
Finally, using relation (1) and the total protein concentration, we obtain a third-degree

polynomial expression for the unknown variable [C]:
PC  C0  Kd2  Kd1 +(Kd2  Kd1 - C0  Kd2 + A0  Kd2 )  C 
(2A 0  Kd2  C0 )  C  C
2
3
(2)
It is easy to check that P(0) = -C0.Kd1.Kd2<0 and that P(∞)=1; hence, it exists at least
 positive real root of the equation P([C])=0 for [C]>0. Let c be this root. Once this
one
value is known, the equilibrium concentrations of the other molecular species are
directly obtained as:
A 
Kd1  Kd2  A0
,
Kd1  Kd2 + Kd2  c + c 2
C  c,
A c ,
M 
(3)
Kd1
D 
A c 2
Kd1  Kd2
.
The unknown parameters of the global problem are the Kd1 dissociation constant, which
is independent of 
the protein interacting with the DNA molecule, and four dissociation
constants, denoted Kd2,LFY-C, Kd2,H387A, Kd2,R390A and Kd2,H387A R390A. For each set of
S9
Supplementary Information
Hamès et al.
experimental data, we derive a distance between the concentration of free DNA, M and
D, as predicted by solving eqs. 2-3, and the experimental counterpart:
EX 
A(C
i1,..., N


2

X, 0,i )  Aexp,i
M(C

2

X0,i )  Mexp,i
i1,..., N
D(C

2
X0,i )  Dexp,i ,
i1,..., N
where X represents one of the proteins under investigation (i.e. LFY-C, H387A,
R390A and H387A R390A); [A]X, [M]X and [D]X are given by eq.3; [A]exp, [M]exp and
[D]exp are the experimental measures of the free DNA and the two DNA-protein
complexes concentrations. Note that the parameter CX0,i, which codes for the ith total
protein concentration (for X= LFY-C, H387A, R390A and H387A R390A) enters the
model through equation (2). Because we consider all experimental data globally, we
determine the best parameter set by minimizing the distance between model and
experiments:
E  E LFYC  E H387A  E R390A  E H387AR390A.
Then, once the best parameter set is determined, the second order variation of the

distance E close to the best fit gives confidence interval for the different parameters and
their ratio.
S10