Tang et al.
Structure of the N-D1 Fragment of p97/VCP mutants
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Supplemental Information
Legends for supplemental figures
Figure S1 Global structure superposition between N-D1 fragments with
bound ATPγS and ADP The Cα trace of the N-D1 structure with ATPγS bound
is shown in the stereoscopic pair in red and that with ADP bound in black. The
N-D1 linker for the ATPγS bound structure is given in cyan and that for ADP
bound is in green.
Figure S2 Structure of R155H mutant p97 N-D1 fragment with bound ADP
(A) The content in the crystallographic asymmetric unit containing two N-D1
hexameric rings is shown for the crystal of the R155H mutant p97 N-D1 fragment
bound with ADP. A view approximately along the six-fold axes of the two rings is
given, showing the two hexameric rings stacked face-to-face with the two ring
axes slightly shifted and a small rotation of one ring with respect to the other
resulting in interdigitating N-domains. (B) Crystal packing environment is shown
for the R155H mutant with ADP bound in the space group P212121. The two
stacked N-D1 rings of p97 in the asymmetric unit are colored in light and deep
blue, respectively.
All symmetry mates are shown in gray of two different
shades. The crystal packing environment precludes the Up-conformation for the
N-domain. (C) The difference electron density for the ADP molecule bound to
the nucleotide-binding site of the R155H mutant p97 is shown in this
stereoscopic pair. The nucleotide-binding site is located at the subunit interface.
1
Tang et al.
Structure of the N-D1 Fragment of p97/VCP mutants
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One subunit is colored in green and the other gray. The ADP is shown as a stick
model with carbon atom in black, oxygen red, nitrogen blue, and phosphorous
orange. It is enclosed in the electron density cage in blue calculated from the
Fourier coefficients of mFo-dFc, where Fc was obtained with ADP omitted, and
contoured at the 3.0σ level.
Figure S3 Translational and rotational movement experienced by the Ndomain from the ADP- to ATPγ S-bound form in N-D1 fragment of p97 The
hexmeric p97 in ATPγS form was aligned with that in ADP form, using only the
D1 domain (rms deviation is 0.692 for 169 residues). The residue G208 was
identified as the point of reference for subsequent calculation, as this residue is
the first residue remaining fixed in position in both forms (point A). The centers
of gravity for N-domains in both conformations were obtained (points B and C).
The translational vector T linking B and C is about 12.5 Å and the angle α is 11°.
To bring the N-domain in the ADP form into superposition with that in ATPγS
form requires a rotation of 92.6° about an axis with polar angles of phi of 77.2°
and psi of 21.4°.
Tables
Table S1. Rms deviations (Å) for pair-wise alignment of different forms of
p97 N-D1 structures.
Table S2. Changes in accessible surface area (ASA) and buried surface
2
Tang et al.
Structure of the N-D1 Fragment of p97/VCP mutants
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area (BSA) at N-D1 interface upon transition from ADP to ATPγS.
3
Tang et al., Figure S1
Tang et al., Figure S2
Tang et al., Figure S3
a
Table S1. Rms deviations (Å) for pair-wise alignment of different forms of p97 N-D1 structures .
N-domain
D1-domain
Wt-ADP
a
(164)
R86A-ATPγS
(171)
R95G-ATPγS
(171)
R155H-ATPγS
(171)
R155H-ADP
(164)
Wt
(ADP)
-
R86A
(ATPγS)
0.691
b
(168)
-
R95G
(ATPγS)
0.665
(161)
0.313
(168)
-
R155H
(ATPγS)
0.739
(169)
0.683
(165)
0.672
(165)
-
R155H
(ADP)
0.842
(159)
1.031
(165)
1.031
(165)
1.031
(165)
-
-
1.490
(215)
-
1.567
(233)
0.324
(263)
-
1.532
(234)
0.503
(258)
0.475
(258)
-
0.915
(255)
1.622
(238)
1.635
(236)
1.635
(236)
-
1.522
(233)
1.562
(222)
0.414
(427)
1.515
(233)
0.698
(439)
0.874
(416)
1.034
(433)
1.678
(239)
1.658
(226)
1.635
(236)
Wt-ADP
(248)
R86A-ATPγS
(248)
R95G-ATPγS
(248)
R155H
(ATPγS)
R155H-ADP
(248)
Wt-ADP
(438)
R86A-ATPγS
(452)
N-D1
R95G-ATPγS
(451)
R155H-ATPγS
(455)
R155H-ADP
(436)
a. Number of residues in the structure.
b. Number of residues aligned.
-
-
-
-
-
Table S2. Changes in accessible surface area (ASA) and buried surface area (BSA) at N-D1 interface upon transition from ADP
to ATPγS.
2
2
Hexamer (Å )
Monomer (Å )
ATPγS (per monomer)
ATPγS
ADP (per monomer)
ADP
a
ASA (N-D1)
116,634 (19,436)
106,124 (17,687)
23,607
20,555
Change in the ASA
+10,510 (+1,752)
+3,052
ASA (D1)
65,591 (9,432)
65,951 (9,432)
14,084
12,949
ASA (N)
64,294 (10,712)
56,996 (9,499)
10,725
9,487
b
BSA at N-D1 interface
13,521 (2,254)
16,607 (2,768)
1,202
1,881
Change in the BSA
-3,356 (559)
-679
BSA with neighboring subunit
6,306 (1,052)
5,321 (887)
Change in BSA with neighboring subunit
+981 (+164)
a. ASA – accessible surface area; BSA – buried surface area.
b. A “+” sign indicates an increase in ASA or BSA upon ATP binding; a “-“ sign indicates a reduction in ASA or BSA.