Missense mutations

The Swiss-pdb Viewer 3.1 program ([57], available on http://www.expasy.org/spdbv/) was used to calculate atomic resolution structural models for SAR1b having missense mutations (Table 3). First, using the 1F6B model [53] and PDB for Cricetulus griseus SAR1b (which lacks the first twelve AA and the 48-55 residues), two residues were modified (I80V, V163I) in order to produce a structural module having a sequence identical to that of human SAR1b. The effects of the missense mutations of AD/CMRD on this "humanized" structure were then modelled.

All the missense mutations are located on the exterior of the three dimensional structure, in strategic places near the recognition, binding and hydrolysis sites for the guanine base (in the N- and C-terminii) and/or affect a highly conserved residue in SAR1/Arf proteins. From the N- to the C- terminus the predicted effects may be summarized as the following (Figure 3). The p.G11D mutation is located in the membrane interacting site (anchorage of the N-terminal part of the molecule) and probably prevents binding to SEC12 and fixation to the ER membrane. The substitution G11P, associated with Y9F and S14F, has been described as being deleterious for vesicle release [38], however no model is available for this mutation (since the coordinates of the first 12 residues of the protein could not be established by the X-ray study leading to the 1F6B structure). The new mutation p.L31P affects the AA just before the active site of GTP and could decrease the GTP hydrolysis. The substitution of a linear (leucine) by a cyclic (proline) residue could lead to steric hindrance (Figure 5). The p.G37R and the p.D75G mutations are located in two different GDP hydrolysis sites. Replacement of glycine 37 by arginine creates steric hindrances with C178 and N134 and the replacement of the aspartic acid 75 by glycine abolishes the H bond with L38. All four of these mutations reduce or eliminate the affinity of SAR1b for GDP/GTP and are expected to

Using the 1F6B model Cricetulus griseus SAR1b [53] and Swiss Pdb Viewer

Figure 5. Localization of missense mutations in the three-dimensional structure of SAR1B

Using the 1F6B model Cricetulus griseus SAR1b [53] and Swiss Pdb Viewer

Figure 5. Localization of missense mutations in the three-dimensional structure of SAR1B

Consequence of

Residue conservation c

PolyPhen prediction (Score)d

SIFT prediction (Score)®

Energy kJ/mol a

Grantham distance b

mutation on prot. (concerned residue) a

Sar1b prot.

Sar1 prot.

Sar1/ Arf family prot.

Small GTP binding prot.

wild type

-9 749

possibly

affect

G11D

modélisation

94

no modelisation

?

0

(0,03)

L31P

-6560

98

steric hindrance (Val97)

+

c

c

c

probably damaging (1,0)

affect protein

(0,02)

G37R

75988

125

steric hindrances (Asn134, Cys178)

+

+

+

(0,00)

E62K

-7777

56

loss of one H-(Glu63)

+

c

+

(0,00)

D75G

-9406

(Lys38)

+

+

+

(0,00)

D137N

-10 086

loss of : one weak H-bond

+

+

+

affect

S179I

-8867

142

(GDP) and one H-bond (Asp137) loss of : one weak H-bond

+

+

affect

S179R

-7550

110

(GDP) and one H-bond (Asp137)

+

(0,00)

L181P

-9023

GDP

c

0

(0,00)

G185 V

127 288

109

Steric hindrance (Met177)

+

+

+

(0,00)

a Swiss Pdb Viewer 3.7 based upon the template 1F6b lacking the first 12 residues of SARlb C.g. (resolution: 1,70a, R value: 0,220, homolgy 98,9%) modified (p.I80V and p.V163I: homology 100% ) b Grantham distance (Alamut ) c http://www.ebi.ac.uk/clustalw/

residue conservation: +, identical; c, conserved substitution; s, semi conserved substitution d PolyPhen-2 v2.2.2r395 http://genetics.bwh.harvard.edu/pph/ e Sorting Intolerant From Tolerant: http://blocks.fhcrc.org/sift/SIFT.html

Table 3. Molecular impact of missense mutations affect the stability of the protein. The substitution p.E62K affecting a well-conserved AA belongs to some residues forming the interface with SEC23 [36], abolishes the H-bond with Glu63 and is predicted to be deleterious by "in silico" analysis (Polyphen, available on http://genetics.bwh.harvard.edu/pph2/ [58] and SIFT available on http://sift.jcvi.org/ [5963]). A H-bond with the guanine in the guanine recognition site is abolished by the p.D137N mutation (Figure 5). Similarly the p.S179I and p.S179R mutations abolish the H-bonds with Asp 137 and with the guanine base. The substitution of a leucine for a proline (L181P) leads to steric hindrance with the guanine base and p.G185V modifies a highly conserved residue in the Arf/Sar1 family and is predicted to be deleterious by "in silico" analysis (Polyphen, SIFT). The last four mutations modify the a helix and p strands in the C-terminus and could affect the stability as well as the conformation of the protein.

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