Table S2. Mutation Effects Predicted by the Structural 3D Analysis
Mutation§
c.592C>T
p.P198S
Description and possible effect
Residue P159 stands on the protein surface in the middle of helix H2 of
domain III. The mutation, introducing a polar residue on the protein
surface, would probably interfere with the correct coupling of GBA with
the protein partners
(P159S)
c.680A>T
p.N227I
(N188I)
c.820G>A
p.E274K
(E235K)
c.850C>A
p.P284T
(P245T)
c.1052G>C
p.W351S
(W312S)
c.1215C>A
p.S405R
(S366R)
R3-1
c.1260G>C
p.W420C
(W381C)
N188 belongs to a loop connecting strand 3 to H3, keeping it folded by
forming hydrogen bonds with the neighbouring loop 240-255 positioned
close to the active site cleft. The replacement with a hydrophobic
isoleucine (N188I) is likely to impact upon the correct substrate
positioning. In addition the introduction of a big, hydrophobic
aminoacid on the protein surface may cause a local misfolding that in
turn could lead to the disruption of the interaction between GBA and its
protein partners
Residue E235 is directly involved in catalysis. Thus the change of an
acidic residue (D) to a basic one (K) is predicted to destroy the catalytic
activity.
Residue P245, is located within the loop 240-255, positioned close to
the active site cleft. The replacement of proline with a threonine, besides
adding some plasticity, would introduce a polar residue in a region that
is mainly hydrophobic, destabilizing the normal shape of the active site
cavity
W312 residue lays at the entrance of the active site cavity. In particular,
the analysis predicted that this aromatic residue may form a 
stacking interaction with the guanidine group of the residue R285.
Comparing the structures of the apo-enzyme to the one of the protein
bound to N-butyl-deoxynojirimycin (NB-DNJ), a chemical chaperone
for GBA, a large displacement of the fragment His 311 – Ala 320
occurs in the presence of NB-DNJ, suggesting that the presence of
W312 and Y313 in this loop is related to a mechanism of substrate
recognition and/or recruitment and that the mutation W312S is
sufficient to destroy this mechanism.
Residue S366, is located in the alpha helix VII close to the active site
cleft. The presence of a large and ramified side chain of the arginine at
this position may destabilize the local fold. Indeed, Ser 366 is hydrogen
bound to W378 promoting stabilization of the beta-sheet encompassing
the active site cleft thus correctly positioning it. Besides the lack of this
hydrogen bond, the mutation of S366R would cause steric collision of
the longer arginine side chain with residues L314, W312, N370.
W381 residue one of seven aromatic side chains flanking the active-site
pocket (19). The introduction of a cysteine in this position not only
might affect catalytic activity, but also might cause a conformational
change by creating a new disulphide bridges. In fact, the in silico
analysis predicted that the newly introduced C in position 381 could
form a disulphide bridge with C18, completely destroying GBA
structure.
Residues N188 and G265 are located on the same region of the protein
surface separated by ca. 20 Å. Both mutations may interfere with the
c.680A>G;c.910G>C
interaction between GBA and other proteins. The replacement of an
asparagine with a serine, a very similar aminoacid in terms of polarity
p.N227S;p.G304R
and size, would have a mild impact on GBA structure. The G265R
mutation introduces a long, ramified and charged side chain in place of
(N188S;G265R)
an hydrogen atom, probably inducing a steric hindrance and the loss of
local main chain plasticity.
E326 residue is located on the protein surface and would be involved
c.1093G>A;c.1255G>A directly in the interaction between the enzyme and Sap-C. Mutation
E326K may interfere with the correct coupling of GBA with protein
p.E365K;p.D419N
partners. Residue N380 is located in the proximity of the W381 residue
one of seven aromatic side chains flanking the active-site pocket. The
N380D mutation may lead to conformational changes perturbing the
(E326K;D380N)
active-site pocket.
§Traditional amino acid residue numbering, which excludes the first 39 aminoacids of the leader
sequence, has been provided in parentheses and designed without the prefix “p.”