Download pH-induced conformational changes in human ABO(H) blood group

Glycobiology vol. 24 no. 3 pp. 237–246, 2014
doi:10.1093/glycob/cwt098
Advance Access publication on November 20, 2013
pH-induced conformational changes in human ABO(H)
blood group glycosyltransferases confirm the importance
of electrostatic interactions in the formation
of the semi-closed state*
Asha R Johal†, Ryan J Blackler†, Javier A Alfaro,
Brock Schuman, Svetlana Borisova, and Stephen V Evans1
Department of Biochemistry and Microbiology, University of Victoria,
Victoria, BC, Canada V8W 3P6
Received on June 21, 2013; revised on October 17, 2013; accepted on
November 6, 2013
The homologous human ABO(H) A and B blood group glycosyltransferases GTA and GTB have two mobile polypeptide loops surrounding their active sites that serve to allow
substrate access and product egress and to recognize and sequester substrates for catalysis. Previous studies have established that these enzymes can move from the “open” state to
the “semi-closed” then “closed” states in response to addition
of a substrate. The contribution of electrostatic interactions
to these conformational changes has now been demonstrated
by the determination at various pH of the structures of GTA,
GTB and the chimeric enzyme ABBA. At near-neutral pH,
GTA displays the closed state in which both mobile loops
order around the active site, whereas ABBA and GTB
display the open state. At low pH, the apparent protonation
of the DXD motif in GTA leads to the expulsion of the donor
analog to yield the open state, whereas at high pH, both
ABBA and GTB form the semi-closed state in which the first
mobile loop becomes an ordered α-helix. Step-wise deprotonation of GTB in increments of 0.5 between pH 6.5 and 10.0
shows that helix ordering is gradual, which indicates that
the formation of the semi-closed state is dependent on
electrostatic forces consistent with the binding of substrate.
Spectropolarimetric studies of the corresponding stand-alone
peptide in solution reveal no tendency toward helix formation
from pH 7.0 to 10.0, which shows that pH-dependent stability
is a product of the larger protein environment and underlines
the importance of substrate in active site ordering.
1
To whom correspondence should be addressed: Tel: +1-250-472-4548; Fax:
+1-250-721-8855; e-mail: [email protected]
*
The atomic coordinates and structure factors (codes 4FRA, 4FRB, 4FRD,
4FRE, 4FRH, 4FRL, 4FRM, 4FRO, 4FRP, 4FRQ, 4FQW, 4GBP and 4KXO)
have been deposited in the Protein Data Bank, Research Collaboratory for
Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://
www.rcsb.org/).
†
A.R.J and R.J.B. are equally contributed.
Keywords: enzyme structure / glycosyltransferases / human
ABO(H) A and B blood groups / pH-induced conformational
change / X-ray crystallography
Introduction
Glycosyltransferases are a diverse group of enzymes (Paulson
and Colley 1989; Sinnott 1990; Campbell et al. 1997;
Charnock and Davies 1999; Unligil and Rini 2000; Bourne and
Henrissat 2001; Breton et al. 2001; Coutinho et al. 2003; Qasba
et al. 2005; Breton et al. 2006; Thiyagarajan et al. 2012) that
synthesize specific carbohydrate structures by catalyzing the
transfer of monosaccharides from an activated donor to an acceptor. Glycosyltransferases fall mainly into two structural families, GT-A and GT-B, which are both excellent examples of the
conservation of a structural phenotype over sequence identity
as there is a high degree of structural similarity even between
enzymes with low sequence homology (Hu and Walker 2002;
Angulo et al. 2006; Letts et al. 2006; Alfaro et al. 2008).
The human ABO(H) blood group A and B glycosyltransferases GTA and GTB (not to be confused with the fold families
GT-A and GT-B) are the most homologous naturally occurring
glycosyltransferases known that utilize distinct naturally occurring donors. These two enzymes execute the final step in the synthesis of the human ABO(H) blood group A and B antigens, where
GTA catalyzes the transfer of N-Acetylgalactosamine (GalNAe)
from (uridine diphosphate) UDP-GalNAc to the H-antigen acceptor [α-L-Fuc-(1→2)-β-D-Gal-OR, where R is a glycoprotein
or a glycolipid] to form the human blood group A antigen and
GTB catalyzes the transfer of galactose (Gal) from UDP-Gal to the
same H-antigen acceptor to form the B antigen (Hearn et al. 1968;
Kobata et al. 1968; Landsteiner 1901; Watkins 1980; Yamamoto
et al. 1990; Patenaude et al. 2002). GTA and GTB differ in only
4 “critical” residues of 354 amino acids: Arg/Gly 176, Gly/Ser
235, Leu/Met 266 and Gly/Ala 268 in GTA and GTB, respectively (Yamamoto et al. 1990; Yamamoto and Hakomori 1990).
The creation of GTA/GTB chimeric enzymes has allowed the
investigation of the influence of each of the four critical residues in donor or acceptor recognition (Yamamoto et al. 1990;
Seto et al. 1995, 1997, 1999, 2000; Palcic et al. 2001; Letts
et al. 2006; Alfaro et al. 2008). These chimeric enzymes are
designated by a four-letter code according to the identity of
© The Author 2013. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
237
AR Johal et al.
each of the four critical residues, where GTA is AAAA and
GTB is BBBB (Seto et al. 1997). As GTA and GTB utilize the
same H-antigen acceptor, their abilities to recognize different
donors were initially ascribed to these critical sequence differences; however, only the last two critical residues (Leu/Met
266 and Gly/Ala 268) were found to impact donor recognition
(Yamamoto and Hakomori 1990; Seto et al. 1997; Kamath
et al. 1999; Patenaude et al. 2002; Marcus et al. 2003; Lee et al.
2005). The first critical residue (Arg/Gly 176) has been shown
to affect enzyme turnover (Lee et al. 2005; Alfaro et al. 2008),
whereas the second critical residue (Gly/Ser 235) influences acceptor recognition (Patenaude et al. 2002; Nguyen et al. 2003;
Letts et al. 2006).
High-resolution structural studies of BBBB, ABBB, AABB
and AAAA revealed two mobile loops adjacent to the active
site: an internal loop consisting of residues 176–188 and a
C-terminal helix formed by residues 346–354 (Patenaude et al.
2002; Letts et al. 2006; Alfaro et al. 2008). These loops display
significant substrate-induced conformational movement (Letts
et al. 2007). Generally, the unliganded enzymes are observed to
lie in an “open” state, where residues in the internal loop are
situated away from the active site or are disordered while the
C-terminal helix is also largely disordered (Alfaro et al. 2008).
This has been attributed to the mutual repulsion of the three
positively charged residues Lys179, Arg180 and Lys124 to destabilize the N terminus of the internal helix, leading to the formation of the open state that allows substrate entry (Letts et al.
2007).
The addition of the charged pyrophosphoryl moiety of the
UDP group appears to counter the repulsion of Lys179, Arg180
and Arg188 and cause the internal mobile loop to order as a
helix over the active site to yield the “semi-closed” state
(Alfaro et al. 2008). The addition of the UDP-sugar donor (or
just UDP) and an acceptor analog can produce the “closed”
state, where the C-terminal loop orders as a helix over the
active site to form interactions with the acceptor L-fucosyl
residue and donor through Lys346, His348 and Arg352 (Alfaro
et al. 2008). The closed state is thought to correspond to the
catalytically active form of the enzyme, as the interactions
between protein and substrate are consistent with the functional
effects of mutagenesis (Angulo et al. 2006; Alfaro et al. 2008;
Shoemaker et al. 2008; Soya et al. 2009).
Interestingly, the identity of the four critical residues affects
the relative stability of the open, semi-closed and closed state
upon substrate binding at near-neutral pH. GTA more readily
assumes the closed state, whereas GTB tends to remain in the
open state, with the chimeric enzymes showing intermediate
behavior correlated to their relative homology to GTA or GTB
(Letts et al. 2006; Alfaro et al. 2008). The same trend is
observed when cryoprotectants mimic the effect of substrate
(Johal et al. 2012). It is interesting to note that the critical residues in the active site of GTB possess larger side chains than
GTA to yield a more sterically crowded active site. However,
the relative stability of the internal loop does not appear to
affect activity strongly, as the two enzymes have almost identical turnover with respect to their cognate substrates (Seto et al.
1997, 1999; Johal et al. 2012).
The activities of many enzymes (including glycosyltransferases) are known to be pH-dependent, with changes in activity
238
usually attributed to such phenomena as changing ionization
states of residues involved in substrate recognition and catalysis
(Perutz 1978; Matthew 1985; Sharp and Honig 1990; Alexov
and Gunner 1997; Jensen 2008). However, manipulating pH to
investigate conformational changes associated with enzyme
action on charged substrates has not been widely explored.
All structures of GTA and GTB reported to date have been
determined within the optimal pH range of activity observed
for these enzymes of pH 6.0–7.6 (Persson and Palcic 2008).
Given the significance of charged-residue interactions between
substrate and protein on the conformations of the active sites of
these enzymes, we have sought to confirm this relationship by
determining the structures of GTA, GTB and the chimeric
enzyme ABBA at various pH values from 5.0 to 10.0.
Results
Data collection and refinement statistics
Data collection and refinement statistics for the enzymes
AAAA and ABBA in complex with UDP and Gal at pH 5.0,
8.0 and 9.0 are given in Table I, whereas data collection and refinement statistics for the wild-type enzyme BBBB at pH 6.5,
7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0 in complex with UDP and
Gal, as well as at pH 10.0 without substrate, are given in
Table II. As loop ordering in these enzymes has already been
Table I. Data collection and refinement results for AAAA and ABBA crystal
structures soaked with UDP and galactose at various pH
pH
Resolution (Å)
Rmerge (%)a,b
Completeness (%)b
Unique reflections
Refinement
Rwork (%)c
Rfree (%)c,d
No. water
r.m.s. bond (Å)e
r.m.s. angle (°)e
B factors (Å2)
Protein
DXDf
Mn2+
PO4 g
Galactose
Occupancy (%)
Mn2+ & PO4
Galactose
PDB ID
ABBA
AAAA
ABBA
ABBA
5.0
20–1.43
2.9 (29.2)
94.8 (91.2)
55,128
5.0
20–2.02
4.7 (30.2)
86.0 (82.4)
17,878
8.0
20–1.54
3.6 (28.7)
98.1 (99.0)
45,812
9.0
20–1.55
3.6 (31.9)
97.8 (99.5)
44,858
18.3
20.8
316
0.027
2.548
19.1
25.8
125
0.019
1.880
18.1
20.5
316
0.026
2.305
17.8
21.4
318
0.025
2.297
19.96
22.20
N/A
19.74
16.71
36.58
34.75
N/A
36.73
33.99
21.15
21.90
22.50
23.39
25.46
24.50
24.10
22.63
25.50
24.23
0 & 20
60
4FRA
0 & 20
30
4FQW
60
80
4FRB
60
80
4FRD
All structures are space group C2221 with unit cell lengths in the range of (a)
52.3–52.67, (b) 149.1–150.0 and (c) 79.3–79.8 Å.
a
Rmerge, Σ|Iobs − Iave|/ΣIave.
b
Values in parentheses represent highest resolution shell.
c
Rwork, Σ||Fo| − |Fc||/Σ|Fo|.
d
10% of reflections were omitted for R-free calculations.
e
r.m.s., root mean square.
f
DXD B-factor is an average of the 4 atoms, Asp211 and Asp213 Oδs, which
coordinate Mn2+.
g
PO4 B-factor is an average of phosphate α- and β-atoms.
pH-induced formation of the semi-closed state in GTB
Table II. Data collection and refinement results for BBBB crystal structures soaked with UDP and galactose at various pH and BBBB at pH 10.0 with no substrate
pH
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.0 (no substrate)
Resolution (Å)
Rmerge (%)a,b
Completeness (%)b
Unique reflections
Refinement
Rwork (%)c
Rfree (%)c,d
No. water
r.m.s. bond (Å)e
r.m.s. angle (°)e
B factors (Å2)
Protein
DXDf
Mn2+
PO4 g
Galactose
Occupancy (%)
Mn2+ & PO4
Galactose
PDB ID
20–1.85
7.2 (31.3)
98.7 (97.7)
25,525
20–1.90
15.8 (21.8)
99.4 (100)
23,426
20–1.80
6.6 (16.4)
99.0 (100)
27,498
20–1.90
8.1 (15.4)
99.7 (100)
23,817
20–2.00
5.9 (13.3)
91.6 (93.1)
18,739
20–1.75
5.1 (16.1)
96.6 (96.5)
29,208
20–2.35
4.4 (8.1)
95.6 (99.1)
12,316
20–2.15
11.3 (48.3)
100 (100)
16,566
20–2.00
7.6 (37.0)
94.9 (98.8)
19,263
18.7
22.2
187
0.021
2.018
17.3
21.8
194
0.020
1.969
17.2
21.2
205
0.021
2.022
18.8
24.0
246
0.019
1.842
18.5
24.8
170
0.018
1.809
18.3
22.1
232
0.021
2.043
18.2
25.2
132
0.016
1.752
16.0
22.5
169
0.018
1.853
23.6
29.0
202
0.018
1.798
23.67
24.61
N/A
27.43
22.57
24.04
22.73
N/A
26.22
22.06
23.25
23.38
19.07
23.00
26.82
24.18
22.03
21.36
21.53
24.05
28.86
25.95
26.56
28.79
27.12
24.32
21.45
22.23
22.68
25.45
24.63
17.10
11.86
15.24
19.72
26.90
20.39
21.60
22.70
22.54
31.64
N/A
N/A
N/A
N/A
0 & 10
50
4FRE
0 & 10
50
4FRM
60
60
4FRH
80 & 60
60
4FRL
80
60
4FRP
80 & 60
60
4FRO
60
60
4FRQ
80
80
4GBP
N/A
N/A
4KXO
All structures are space group C2221 with unit cell lengths in the range of (a) 52.1–52.7, (b) 150.0–151.4 and (c) 79.0–79.5 Å.
Rmerge, Σ|Iobs − Iave|/ΣIave.
b
Values in parentheses represent highest resolution shell.
c
Rwork, Σ||Fo| − |Fc||/Σ|Fo|.
d
10% of reflections were omitted for Rfree calculations.
e
r.m.s., root mean square.
f
DXD B-factor is an average of the 4 atoms, Asp211 and Asp213 Oδs, which coordinate Mn2+.
g
PO4 B-factor is an average of phosphate α- and β-atoms.
a
Table III Effect of pH on loop ordering in AAAA, ABBA and BBBB
Enzyme
AAAA+UDP+Gal
ABBA+UDP+Gal
BBBB+UDP+Gal
BBBB
pH
Acceptor Donor site
site
5.0
30%
6.5a 100%
5.0
60%
6.5a 100%
8.0
80%
9.0
80%
6.5
50%
7.0
50%
7.5
60%
8.0
60%
8.5
60%
9.0
60%
9.5
60%
10.0
80%
10.0
-
Water
Water
Water
Water
Water
Water
Water
Water
Water
Water
Water
Water
Water
Gal
-
UDP site
Mn2+
site
Weak
0%
Weak PO4
100%
Weak
0%
Fragmented 40%
Good
60%
Good
60%
Fragmented
0%
Fragmented
0%
Good
60%
Good
80%
Good
80%
Good
80%
Good
60%
Good
80%
-
Internal loop
EV
EV
EV
EV
EV
EV
EV
EV
EV
EV
EV
EV
EV
EV
EV
C-terminus
176
181
186
191
196
Raykr
RAYKR
Raykr
RAykr
RAYKr
RAYKR
Gaykr
Gaykr
Gaykr
GAYKR
GAYKR
GAYKR
GAYKR
GAYKR
Gaykr
wqdvs
WQDVS
wqdvs
wqdvs
wqDVS
WQDVS
wqdvs
wqdvs
wQDVS
WQDVS
WQDVS
WQDVS
WQDVS
WQDVS
wqdvS
m RRME
M RRME
m RRME
M RRME
M RRME
M RRME
m RRME
m RRME
M RRME
M RRME
M RRME
M RRME
M RRME
M RRME
M RRME
MISdf
MISdf
MISdf
MISdf
MISdf
MISdf
MISdf
MISdf
MISdf
MISdf
MISdf
MISdf
MISdf
MISDF
MISDF
CER R
CER R
CER R
CER R
CER R
CER R
CER R
CER R
CER R
CER R
CER R
CER R
CER R
CER R
CER R
VP
VP
VP
VP
VP
VP
VP
VP
VP
VP
VP
VP
VP
VP
VP
346
351
knhqa
KNHQA
knhqa
knhqa
KNhqa
knhqa
knhqa
knhqa
Knhqa
KNhqa
knhqa
Knhqa
KNhqa
KNhqa
knhqa
vr np
VRNP
vr np
vr np
vr np
vr np
vr np
vr np
vr np
vr np
vr np
vr np
vr np
vr np
vr np
Black one letter amino acid codes correspond to electron density for main chain and side chain atoms, green/italic upper case letters correspond to electron density
for main chain atoms only and red/underlined upper case letters correspond to weak electron density for main and side chain atoms. Residues with red/lower case
one letter amino acid codes have not been included in the refined models.
a
Johal et al. 2012
shown to be sensitive to the choice of glycerol as a cryoprotectant, the structures were all collected using 20% 2-methyl2,4-pentanediol (MPD) (v/v).
A summary of the observed electron density surrounding
the internal mobile loop (residues 176–188) and the C-terminal
loop (residues 346–354) for all complexes is shown in Table III,
which includes the reported structures of AAAA and ABBA at
pH 6.5 (Johal et al. 2012) that lie within the optimal pH range of
6.0–7.6 for GTA and GTB (Persson and Palcic 2008). Excellent
density for almost the entire length of the remaining polypeptide
is seen in all structures, with no other pH-induced structural
changes observed. In all structures presented here, the “open
state” refers to the lack of observed electron density for internal
loop residues 176–186.
239
AR Johal et al.
Wild-type AAAA and chimeric ABBA structures lie in the
open state at lower pH values and the semi-closed state at
higher pH values (Table III). The structures of wild-type
BBBB + UDP + Gal at carefully adjusted values of pH in intervals of 0.5 from pH 6.5 to pH 10.0 display a gradual change in
state from the open state at lower pH to the semi-closed state at
higher pH, whereas BBBB with no substrate at pH 10.0 displays the open state (Table III).
Substrate binding
All substrate soaking trials used UDP as a donor analog. To minimize the influence of the C-terminal loop on this study of the internal loop, all enzymes (except the structure of BBBB with no
substrates at pH 10.0) were crystallized in the presence of galactose as an H-antigen acceptor analog. The formation of the closed
state in which the C-terminal loop is ordered is strongly affected
by contact from the C-terminal loop to the L-fucose residue of the
H-antigen acceptor (Alfaro et al. 2008). Accordingly, with the
exception of GTA, which has recently been shown to form
the closed state most readily even in the absence of the acceptor
fucose moiety (Johal et al. 2012; Alfaro et al. 2008), the
C-terminal loop is not ordered in any of the structures with
simple galactose as an acceptor analog at any pH. ABBA was
chosen for this study as it was known to have loop stability
intermediate between GTA and GTB (Johal et al. 2012).
In all 12 structures to which galactose was added regardless
of the pH, a galactose molecule is present in the acceptorbinding site. UDP is also bound to the donor-binding site, although the pyrophosphoryl moiety is sometimes disordered
(Table III). In the chimeric ABBA structures, galactose displayed appropriate electron density but refined to a slightly
higher occupancy at pH 5.0 than at pH 8.0 and pH 9.0. In contrast, UDP was observed with weakest electron density at pH
5.0. The substrate analogs in BBBB structures had generally
constant occupancies and quality of electron density over the
pH range tested, with the exception of the pH 6.5 and 7.0 structures that had fragmented UDP density (Table III). At pH 10.0,
a second galactose molecule was observed with good electron
density in the donor site where the galactosyl residue of
UDP-Gal would be expected to bind. Unless otherwise noted,
the positions and interactions of UDP and Gal are consistent
with those previously reported for these enzymes. Ordered
MPD molecules were not observed in any structure. An ordered
polyethylene glycol (PEG) is observed near His140 and His219
on the surface of the ABBA and AAAA structures at pH 5.0.
PEG is observed in this location in the BBBB + UDP + Gal structures at pH 6.5, 7.5 and 9.5 and a glycine molecule at pH 9.0.
Spectropolarimetric studies at various pH
To test if the pH-dependent ordering of the internal loop of GTB
was an intrinsic property of the primary structure of the loophelix spectropolarimetry (circular dichroism, CD) was used to investigate the helicity of the peptide corresponding to residues
Tyr178-Asp194 in solution at various pH. Mean residue ellipticity for the peptide at pH 7.0, 8.0, 9.0 and 10.0 are shown in
Supplementary data, Figure S1. The spectra do not reveal characteristic peaks of a regular secondary structure, but possess a
strong negative peak near 197 nm consistent with unordered
oligopeptide as observed in benchmark studies by Davidson,
240
Greenfield and Fasman of the optical rotary dispersion of
poly-L-lysine (Davidson and Fasman 1967; Greenfield et al.
1967; Greenfield and Fasman 1969). Specifically, there is no
characteristic positive peak near 192 nm nor negative peaks near
208 and 222 nm that correspond to an α-helix secondary structure (Juban et al. 1997; Greenfield 2006). These observations are
consistent with results from secondary structure predictions on
DichroWeb server using larger reference datasets (van Stokkum
et al. 1990); predictions were between 8.5 and 16.5% helicity for
the four pH conditions examined (Supplementary data, Table SI).
Discussion
Low pH yields the open state in GTA
At neutral pH in the presence of UDP and galactose, GTA is
observed to lie in the closed state, whereas both GTB and
ABBA lie in the open state, with ordered UDP, galactose and
manganese ion in all three. This differential behavior has been
noted before and generally follows the pattern GTA > chimera >
GTB will more readily form the semi-closed state (Alfaro et al.
2008; Johal et al. 2012).
GTA so easily forms the semi-closed and the closed state that
it has not previously been observed in the open state; however,
at pH 5.0, both AAAA and ABBA lie in the open state. The
reason is immediately apparent as neither structure shows appropriate electron density corresponding to the UDP group or
manganese ion that normally coordinates to the DXD motif and
the pyrophosphoryl moiety of the UDP. The absence of the
manganese ion is probably due to the protonation of the aspartate residues of the DXD motif, which renders the enzyme incapable of effectively binding UDP (Table III).
The formation of the semi-closed and closed states has been
attributed to the ability of the negatively charged UDP moiety
to counter the positively charged residues on the active site
loops (Letts et al. 2007; Figure 1), so the display of the open
state in these enzymes at low pH is consistent with the loss of
the electrostatic contribution of UDP.
Higher pH yields the semi-closed state in GTB and ABBA
Structures of the chimeric enzyme ABBA + UDP + Gal are
observed in the open state at pH 5.0 and 6.5 (Alfaro et al. 2008;
Johal et al. 2012) and the semi-closed state at pH 9.0
(Table III). Similarly, BBBB + UDP + Gal lies in the open state
at neutral pH and transitions to the semi-closed state as the pH
increases. These higher pH structures display the same general
conformations and interactions observed in the substrate-induced
closed and semi-closed structures seen for AABB at neutral pH
(Alfaro et al. 2008), specifically: Arg180-Glu297, Arg187-Glu190,
Arg188-Asp302, Arg188-Asp211, Arg199-Glu203 and Lys346Asp213, with the manganese ion coordinated to the DXD motif
and to the pyrophosphoryl moiety of UDP.
The mutual repulsion of positively charged residues on the
internal mobile loop and on the lip of the active site had been
postulated to yield the open state in GTB and chimeric
enzymes (Letts et al. 2007; Alfaro et al. 2008). These enzymes
undergo a conformational shift to the semi-closed state
upon the introduction of the negatively charged UDP-donor
(Figure 1), which offsets the positive charges and forms a
number of specific interactions with the protein.
pH-induced formation of the semi-closed state in GTB
Fig. 1. Influence of charged residue interactions on the mobility of the internal and C-terminal loops. In the unliganded ABBB structure (light, PDB 2RIZ), the
presence of nearby Lys124 creates a mutual repulsion with internal loop residues Lys179 and Arg180 to destabilize the enzyme and form the open state. Addition of
uridine diphosphate (UDP) as seen in ABBB + UDP + HA (dark, PDB 2RJ1) displays the negatively charged pyrophosphoryl groups on UDP to overcome the
mutual repulsion created by the positive charges and form the semi-closed state.
The “structural titration” of GTB in the current study gives
direct support to this hypothesis, as the higher pH has the effect
of lessening the mutual repulsion between the positively
charged residues (Jancan and Macnaughtan 2012) and so
allows the semi-closed state to form. There would be no deprotonation expected in the active site until approximately pH 9.4–
10.0, where the imide moiety of UDP is ionized (McElroy
1951; Bock et al. 1956; Dawson et al. 1969; Jancan and
Macnaughtan 2012) to yield a stabilizing negative charge.
It is interesting to note that the stabilization of the semiclosed state in GTB induced by high pH is not sufficient to
offset the contribution made by binding UDP, as the structure
without substrate at pH 10.0 displays the open state (Table III).
pH-induced loop ordering in GTB is gradual
The relative instability of GTB toward the semi-closed state
allowed the examination of the effect of incremental changes of
pH on loop ordering. Separate crystals of BBBB + UDP + Gal
soaked in increasingly basic mother liquor in increments of 0.5
pH units from 6.5 to 10.0 display a gradual improvement of
electron density for the internal mobile loop, ranging from a
broad lack of electron density at pH 6.5 to complete main chain
density and most side chain density at pH 10.0 (Figure 2).
Superposition of the structure of BBBB + UDP + Gal at pH
10.0 with substrate-bound chimeric structures at neutral pH
shows that the backbone of the internal loop of GTB at higher
pH has adopted the same conformation as that induced by substrate analogs in the chimeric enzymes (Alfaro et al. 2008).
Interestingly, the ordering of the loop is gradual, i.e. it does
not happen upon the deprotonation of a side chain passing its
pKa. This is supported by the calculation of ionization states of
residues in the mobile loop over this pH range using PROPKA
(Li et al. 2005; Bas et al. 2008; Olsson et al. 2011; Sondergaard
et al. 2011), which predicts that there are no residues in the
vicinity of the mobile loop with pKa values in the pH range
under study. Instead, it appears that the gradual improvement of
density is due to a broad stabilization of charged-residue interactions with increasing pH (Wood 1974). For example, there
would be a decreasing repulsion between Lys179 N-terminal to
the helix and Lys124 near the lip of the active site ( predicted
pKa: Lys179 = 10.33; Lys124 = 9.93), and a similar effect involving Lys179, Arg180 and the partial positive charge of the
helix dipole N terminus.
Although at the final pH of 10.0 some of the surface exposed
lysine residues would be expected to be partially deprotonated,
the electron density for the main chain and side chain of
Lys124 is greatly improved over the corresponding residue in
lower pH structures as it forms hydrogen bonds to the main
chain oxygen of both Tyr178 and Lys179. Main chain density
for the latter two residues was fragmented in lower pH structures but is also improved at pH 10.0. As well, the side chain of
Lys346 displayed poor density at lower pH, but is fully ordered
at pH 10.0 where it forms a salt bridge to an oxygen atom on
the pyrophosphoryl moiety of UDP.
The gradual ordering of GTB stands in contrast to reported
examples of proteins that exhibit a significant conformational
change over a narrow pH range. The Bombyx mori PheromoneBinding-Protein (BmorPBP) flips between an “open” and a
“closed” state below and above pH 5.5, respectively, which has
been hypothesized to depend on the protonation state of three
conserved histidine residues (Lautenschlager et al. 2005). Other
known examples are bovine β-lactoglobulin, which displays the
opening of an active site “lid” loop and a change in the dimer
interface somewhere between pH 6.0 and 8.0 (Qin et al. 1998),
and the well-characterized human prion that undergoes reversible (and eventually irreversible) folding at acidic pH
(Swietnicki et al. 1997). The mutant glycinamide ribonucleotide
241
AR Johal et al.
Fig. 2. Stereo views of the 2Fo-Fc electron density maps contoured at 1.0 σ for the internal loop in GTB structures from pH 6.5 to 10.0 show a gradual appearance of
electron density corresponding to the semi-closed state as the solution becomes more basic.
transformylase from Escherichia coli displays ordering of an
active site loop helix between crystal structures at pH 3.5 and 7.5
(Su et al. 1998); however, the lack of structures at intermediate
pH values precludes a direct comparison.
The study was not continued past pH 10.0 as a precipitate
consistent with the formation of Mn(OH)2 was formed. Given a
Ksp of 2.0 × 10−13 (Fox et al. 1941), 5 mM Mn2+ would be
expected to precipitate as Mn(OH)2 at approximately pH 10.3.
242
Spectropolarimetry confirms the primary influence
of the protein
CD has demonstrated that the structures of particular α-helix
segments can be pH-dependent (for a review, see Munoz and
Serrano 1995). The factors shown to be foremost are the arrangement of positive and negative side-chains and the proximity of charges to the macrodipole N- and C-termini. The helix
within the mobile loop of GTA and GTB contains a number of
pH-induced formation of the semi-closed state in GTB
charged residues (Arg180, Asp183, Arg187, Arg188 and
Glu190) that indicated potential susceptibility to short-range
electrostatic forces and that the increasing tendency of this
segment to form a helix with increasing pH could be intrinsic.
However, spectropolarimetic studies of the secondary structure of the peptide of corresponding sequence revealed no
significant change in mean residue ellipticity at 222 nm,
which indicates that there is no change in helicity with pH
(Supplementary data, Fig. S1 and Table SI). Indeed, the results
are not characteristic of any regular secondary structure over the
pH range tested, which provides strong evidence that the ordering of the internal loop is dependent on the electrostatic and
steric environment of the enzyme active site.
It is well established that the local charge environment can
affect the electrostatic potential of a functional group. For
example, the pI of GTB is calculated to be 9.0 based on its tertiary structure (Li et al. 2005; Bas et al. 2008; Olsson et al.
2011; Sondergaard et al. 2011), but 8.3 based solely on sequence (using ProtParam, ExPASy; Gasteiger et al. 2003).
Analysis of the observed structure of the internal loop helix
with PROPKA (Li et al. 2005; Bas et al. 2008; Olsson et al.
2011; Sondergaard et al. 2011) predicts that this helix should
experience marginally increasing stability with pH to a
maximum at pH 10.0–11.0. The calculated free energy of stabilization going from pH 7.0 to 10.0 of only −0.3 kcal/mol corroborates the failure to observe corresponding structures for the
peptide in solution.
GTB at pH 10.0 contains a galactose molecule
in the donor-binding site
The structure of BBBB + UDP + Gal at pH 10.0 has an additional galactose molecule in the donor sugar binding site (occupied by water molecules in corresponding structures at lower
pH) in addition to a galactose molecule that is found in the acceptor site of all structures. Interestingly, this corresponds to the
previously published structure of AAAA + UDP + Gal at pH
7.0 (Johal et al. 2012), where the galactose was observed to
form the same interactions with His301, Asp302, Arg188 and
Asp211 with the enzyme in the semi-closed state.
Conclusion
This series of structures at increasing pH is a unique structural
characterization of gradual peptide-loop ordering and emphasizes the highly sensitive structural and electrostatic environment that affects the specificity and activity of these enzymes.
The induction of the semi-closed state in GTB and the chimeric enzyme ABBA with increasing pH demonstrates that the
formation of the catalytically competent conformation of the
enzyme is promoted by electrostatic interactions with a substrate. Just as binding of the negatively charged UDP-sugar
donor would offset local positive charges (Alfaro et al. 2008;
Johal et al. 2012), increasing pH would lower and eventually
neutralize their mutual repulsion.
The reported active pH range of most glycosyltransferases,
including GTA and GTB, of 5.5–7.0 is consistent with the environment of the ER-Golgi plasmalemma. The structures of
GTA, ABBA and GTB show a clear rationale for the inactivity
of the enzymes at low pH, as the protonation of the active site
DXD motif and concomitant expulsion of the Mn2+ cofactor is
incompatible with catalysis. However, the upper limit of activity is well below the pH that would affect the dynamics of the
internal loop of GTB.
Materials and methods
Crystallization
The chimeric ABBA protein was crystallized in protein stock
solution as described in Alfaro et al. (2008). Crystals of GTA
and GTB were grown at 4°C from a concentration of protein
(30–40 mg/mL for GTB and 16–20 mg/mL for GTA) along
with 1% PEG 4000, 4.5–5% MPD, 100 mM ammonium sulfate,
70 mM sodium chloride, 50 mM N-[2-acetamido]-2-iminodiacetic
acid (ADA) buffer pH 7.5, 30 mM sodium acetate buffer pH 4.6
and 5 mM MnCl2.
Crystals of ABBA and GTA were placed against a reservoir
containing 3.7% PEG 4000, 7% MPD, 0.3 M ammonium
sulfate, 0.25 M sodium chloride, 0.2 M ADA buffer and 0.1 M
sodium acetate. The crystals usually grew to 0.3 mm in width
over 5–10 days at 4°C. Before making complexes, ABBA and
GTA crystals were washed with artificial mother liquor containing 3.5% PEG 4000, 50 mM ammonium sulfate, 40 mM
sodium chloride, 35 mM ADA buffer and 15% MPD. As the
structures of these enzymes are known to be sensitive to cryoprotectant identity, crystals of AAAA and ABBA in complex
with UDP and galactose were obtained by placing them in
mother liquor with 15% MPD, 50 mM UDP, 150 mM galactose
and 5 mM MnCl2 for 2–5 days at 4°C. All substrate analogs were
added incrementally over a period of a few hours so as to prevent
crystal fracture. The chimeric structure of ABBA + UDP + Gal
was examined at pH 5.0, 8.0 and 9.0, and the wild-type structure
of AAAA + UDP + Gal was examined at pH 5.0.
Crystals of GTB with UDP and galactose in pH increments of
0.5 from 6.5 to 10.0 were obtained by soaking crystals of GTB in
crystallization solution with the addition of 15% MPD, 5 mM
MnCl2, 50 mM UDP and 150 mM galactose and with buffer
replaced by the following: ADA pH 6.5, Tris–HCl pH 7.0, 7.5,
8.0 and 8.5 and glycine pH 9.0, 9.5 and 10.0. The final pH of
each solution was adjusted with NaOH to match that of the buffer
used and tested with pH paper strips from EMD Chemicals (sensitive to 0.2 pH units). The structure of GTB at pH 10.0
without substrate was collected from a crystal of GTB washed in
crystallization solution with Ethylenediaminetetraacetic acid to
remove residual MnCl2 and UDP and then soaked in crystallization solution with glycine pH 10.0.
Data collection, reduction and structure determination
X-ray diffraction data were collected at −160°C for all crystals
using a CryoStream 700 crystal cooler. Before freezing the
crystals for data collection, the concentration of the cryoprotectant was made by replacing a corresponding volume of water
with 20% MPD (v/v). For the ABBA and AAAA structures,
X-ray diffraction data were collected on a Rigaku R-AXIS IV+
+ area detector at distances of 72 mm and exposure times
between 4.0 and 7.0 min for 0.5° oscillations. X-rays were produced by an MM-002 generator (Rigaku Americas, College
Station, TX) coupled to Osmic “Blue” confocal X-ray mirrors
with power levels of 30 W (Osmic, Auburn Hills, MI). The
243
AR Johal et al.
data were scaled, averaged and integrated using d*trek and
CrystalClear (Pflugrath 1999). For the BBBB structures with
pH ranging from 6.5 to 9.5, X-ray diffraction data were collected at the Canadian Macromolecular Crystallography
Facility on beamline 08ID-1 (CMCF-ID) of the Canadian Light
Source (Grochulski et al. 2011) and processed using HKL2000
(Otwinowski and Minor 1997).
For the BBBB structures at pH 10.0, X-ray diffraction data
were collected on a Rigaku R-AXIS IV++ area detector at a distance of 72 mm and exposure time of 5.0 min for 0.5° oscillations. X-rays were produced by an MM-003 generator (Rigaku
Americas) coupled to Osmic “Blue” confocal X-ray mirrors with
power levels of 30 W (Osmic). The data were scaled, averaged
and integrated using HKL2000 (Otwinowski and Minor 1997).
Although the structures were expected to be nearly isomorphous, for completeness all structures were solved by molecular replacement using the CCP4 module Phaser (McCoy
et al. 2007; Winn et al. 2011) with a previously solved GTA
structure in the “closed” conformation as a starting model
(PDB accession code 3SXG). For ABBA and AAAA structures, model building and refinement were carried out with the
CCP4 module REFMAC5 (Murshudov et al. 2011; Winn et al.
2011), SetoRibbon (Evans, unpublished) and Phenix (Adams
et al. 2002). For BBBB structures, model building and refinement were carried out with Coot (Emsley and Cowtan 2004),
SetoRibbon (Evans, unpublished), CCP4 module REFMAC5
(Murshudov et al. 2011; Winn et al. 2011) and Phenix (Adams
et al. 2002). All figures were produced using SetoRibbon
(Evans, unpublished).
Careful note was made during the refinement of the isotropic
temperature factors of the Mn2+ ion and the UDP substrates,
with care taken to adjust occupancies in increments of 10%
such that the temperature factors were comparable with those of
the side-chain oxygen atoms of the DXD motifs to which they
were associated (Table I).
Spectropolarimetry
Peptide corresponding to amino acid residues Tyr178-Asp194
of the internal mobile loop helix was purchased synthesized
from GenScript with N-terminal acetylation and C-terminal
amidation (Ac-YKRWQDVSMRRMEMISD-NH2). Peptide
was dissolved in CD buffer (1 mM sodium citrate, 1 mM
sodium phosphate, 1 mM sodium borate) at pH 7.0, 8.0, 9.0
and 10.0 to a final concentration of 0.1 mg/mL. CD spectra
from 185 to 260 nm were acquired at room temperature in a
quartz cell of 1 mm path length on a Jasco-J720 spectropolarimeter, using a continuous scanning mode with a wavelength
interval of 0.5 nm, scan speed of 50 nm/min, response time of
1.0 s and band width of 1.0 nm. Spectra at each pH were collected in triplicate and averaged, and spectra of buffer blanks
were subtracted. Data were processed using the application
CONTIN (van Stokkum et al. 1990) on DichroWeb (Whitmore
and Wallace 2008) to generate mean residue ellipticities and
secondary structure predictions.
Supplementary Data
Supplementary data for this article is available online at http://
glycob.oxfordjournals.org/.
244
Funding
This work was supported by a grant from the Canadian
Institutes of Health Research to SVE. SVE is a recipient of a
Michael Smith Foundation for Health Research Senior
Scholarship. RJB is a recipient of a Natural Sciences and
Engineering Research Council of Canada CGS award.
Acknowledgements
We thank Professor Juan Ausio for generously allowing the use
of his spectropolarimeter for the CD studies described in this
paper.
Conflict of interest
None declared.
Abbreviations
ADA, N-[2-acetamido]-2-iminodiacetic acid; CD, circular dichroism; GAL, galactose; GTA, human ABO(H) blood group A
α-1,3-N-acetylgalactosaminyltransferase; GTB, human ABO(H)
blood group B α-1,3-galactosyltransferase; MPD, 2-methyl-2,4pentanediol; PEG, polyethylene glycol; UDP, uridine diphosphate.
References
Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, Moriarty
NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC. 2002. PHENIX:
Building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr. 58:1948–1954.
Alexov EG, Gunner MR. 1997. Incorporating protein conformational flexibility
into the calculation of pH-dependent protein properties. Biophys J.
72:2075–2093.
Alfaro JA, Zheng RB, Persson M, Letts JA, Polakowski R, Bai Y, Borisova SN,
Seto NO, Lowary TL, Palcic MM, et al. 2008. ABO(H) blood group A and
B glycosyltransferases recognize substrate via specific conformational
changes. J Biol Chem. 283:10097–10108.
Angulo J, Langpap B, Blume A, Biet T, Meyer B, Krishna NR, Peters H, Palcic
MM, Peters T. 2006. Blood group B galactosyltransferase: Insights into substrate binding from NMR experiments. J Am Chem Soc. 128:13529–13538.
Bas DC, Rogers DM, Jensen JH. 2008. Very fast prediction and rationalization
of pK(a) values for protein-ligand complexes. Proteins Struct Func
Bioinform. 73:765–783.
Bock RM, Ling NS, Morell SA, Lipton SH. 1956. Ultraviolet absorption
spectra of adenosine-5′-triphosphate and related 5′-ribonucleotides. Arch
Biochem Biophys. 62:253–264.
Bourne Y, Henrissat B. 2001. Glycoside hydrolases and glycosyltransferases:
Families and functional modules. Curr Opin Struct Biol. 11:593–600.
Breton C, Mucha J, Jeanneau C. 2001. Structural and functional features of glycosyltransferases. Biochimie. 83:713–718.
Breton C, Snajdrova L, Jeanneau C, Koca J, Imberty A. 2006. Structures and
mechanisms of glycosyltransferases. Glycobiology. 16:29R–37R.
Campbell JA, Davies GJ, Bulone V, Henrissat B. 1997. A classification of
nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochem J. 326 (Pt 3):929–939.
Charnock SJ, Davies GJ. 1999. Structure of the nucleotide-diphospho-sugar
transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed
forms. Biochemistry. 38:6380–6385.
Coutinho PM, Deleury E, Davies GJ, Henrissat B. 2003. An evolving hierarchical family classification for glycosyltransferases. J Mol Biol. 328:307–317.
Davidson B, Fasman GD. 1967. The conformational transitions of uncharged
poly-L-lysine. Alpha helix-random coil-beta structure. Biochemistry.
6:1616–1629.
Dawson RMC, Elliott DC, Elliott WH, Jones KM. 1969. Data for Biochemical
Research. New York: Oxford University Press.
pH-induced formation of the semi-closed state in GTB
Emsley P, Cowtan K. 2004. Coot: Model-building tools for molecular graphics.
Acta Crystallogr D Biol Crystallogr. 60:2126–2132.
Fox RK, Swinehart DF, Garrett AB. 1941. The equilibria of manganese hydroxide, Mn(OH)(2), in solutions of hydrochloric acid and sodium hydroxide.
J Am Chem Soc. 63:1779–1782.
Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A. 2003.
ExPASy: The proteomics server for in-depth protein knowledge and analysis.
Nucleic Acids Res. 31:3784–3788.
Greenfield N, Davidson B, Fasman GD. 1967. The use of computed optical rotatory dispersion curves for the evaluation of protein conformation.
Biochemistry. 6:1630–1637.
Greenfield N, Fasman GD. 1969. Computed circular dichroism spectra for the
evaluation of protein conformation. Biochemistry. 8:4108–4116.
Greenfield NJ. 2006. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc. 1:2876–2890.
Grochulski P, Fodje MN, Gorin J, Labiuk SL, Berg R. 2011. Beamline 08ID-1,
the prime beamline of the Canadian Macromolecular Crystallography
Facility. J Synchrotron Radiat. 18:681–684.
Hearn VM, Smith ZG, Watkins WM. 1968. An a-N-acetyl-Dgalactosaminyltransferase associated with the human blood-group A character. Biochem J. 109:315–317.
Hu Y, Walker S. 2002. Remarkable structural similarities between diverse glycosyltransferases. Chem Biol. 9:1287–1296.
Jancan I, Macnaughtan MA. 2012. Acid dissociation constants of uridine-5′diphosphate compounds determined by 31phosphorus nuclear magnetic resonance spectroscopy and internal pH referencing. Anal Chim Acta.
749:63–69.
Jensen JH. 2008. Calculating pH and salt dependence of protein-protein
binding. Curr Pharm Biotech. 9:96–102.
Johal AR, Schuman B, Alfaro JA, Borisova S, Seto NOL, Evans SV. 2012.
Sequence-dependent effects of cryoprotectants on the active sites of the
human ABO(H) blood group A and B glycosyltransferases. Acta Crystallogr
D Biol Crystallogr. 68:268–276.
Juban MM, Javadpour MM, Barkley MD. 1997. Circular dichroism studies of
secondary structure of peptides. Methods Mol Biol. 78:73–78.
Kamath VP, Seto NO, Compston CA, Hindsgaul O, Palcic MM. 1999.
Synthesis of the acceptor analog alphaFuc(1–>2)alphaGal-O(CH2)7 CH3: A
probe for the kinetic mechanism of recombinant human blood group B glycosyltransferase. Glycoconj J. 16:599–606.
Kobata A, Grollman EF, Ginsburg V. 1968. An enzymatic basis for blood type
B in humans. Biochem Biophys Res Commun. 32:272–277.
Landsteiner K. 1901. Über Agglutinationserscheinungen normalen menschlichen Blutes. Wien Klin Wochenschr. 14:1132–1134.
Lautenschlager C, Leal WS, Clardy J. 2005. Coil-to-helix transition and ligand
release of Bombyx mori pheromone-binding protein. Biochem Biophys Res
Commun. 335:1044–1050.
Lee HJ, Barry CH, Borisova SN, Seto NO, Zheng RB, Blancher A, Evans SV,
Palcic MM. 2005. Structural basis for the inactivity of human blood group
O2 glycosyltransferase. J Biol Chem. 280:525–529.
Letts JA, Persson M, Schuman B, Borisova SN, Palcic MM, Evans SV. 2007.
The effect of heavy atoms on the conformation of the active-site polypeptide
loop in human ABO(H) blood-group glycosyltransferase B. Acta Crystallogr
D Biol Crystallogr. 63:860–865.
Letts JA, Rose NL, Fang YR, Barry CH, Borisova SN, Seto NO, Palcic MM,
Evans SV. 2006. Differential recognition of the type I and II H antigen acceptors by the human ABO(H) blood group A and B glycosyltransferases. J Biol
Chem. 281:3625–3632.
Li H, Robertson AD, Jensen JH. 2005. Very fast empirical prediction and rationalization of protein pKa values. Proteins. 61:704–721.
Marcus SL, Polakowski R, Seto NO, Leinala E, Borisova S, Blancher A,
Roubinet F, Evans SV, Palcic MM. 2003. A single point mutation reverses
the donor specificity of human blood group B-synthesizing galactosyltransferase. J Biol Chem. 278:12403–12405.
Matthew JB. 1985. Electrostatic effects in proteins. Annu Rev Biophys Biophys
Chem. 14:387–417.
McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read
RJ. 2007. Phaser crystallographic software. J Appl Crystallogr. 40:658–674.
McElroy WDGB. 1951. Phosphorus Metabolism. Baltimore: John Hopkins
University Press.
Munoz V, Serrano L. 1995. Elucidating the folding problem of helical peptides
using empirical parameters. III. Temperature and pH dependence. J Mol
Biol. 245:297–308.
Murshudov GN, Skubak P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA,
Winn MD, Long F, Vagin AA. 2011. REFMAC5 for the refinement of
macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr.
67:355–367.
Nguyen HP, Seto NO, Cai Y, Leinala EK, Borisova SN, Palcic MM, Evans SV.
2003. The influence of an intramolecular hydrogen bond in differential recognition of inhibitory acceptor analogs by human ABO(H) blood group A
and B glycosyltransferases. J Biol Chem. 278:49191–49195.
Olsson MHM, Sondergaard CR, Rostkowski M, Jensen JH. 2011. PROPKA3:
Consistent treatment of internal and surface residues in empirical pK(a) predictions. J Chem Theory Comput. 7:525–537.
Otwinowski Z, Minor W. 1997. Processing of X-ray diffraction data collected in
oscillation mode. Macromol Crystallogr A. 276:307–326.
Palcic MM, Seto NO, Hindsgaul O. 2001. Natural and recombinant A and B
gene encoded glycosyltransferases. Transfus Med. 11:315–323.
Patenaude SI, Seto NO, Borisova SN, Szpacenko A, Marcus SL, Palcic MM,
Evans SV. 2002. The structural basis for specificity in human ABO(H) blood
group biosynthesis. Nat Struct Biol. 9:685–690.
Paulson JC, Colley KJ. 1989. Glycosyltransferases. Structure, localization, and
control of cell type-specific glycosylation. J Biol Chem. 264:17615–17618.
Persson M, Palcic MM. 2008. A high-throughput pH indicator assay for screening glycosyltransferase saturation mutagenesis libraries. Anal Biochem.
378:1–7.
Perutz MF. 1978. Electrostatic effects in proteins. Science. 201:1187–1191.
Pflugrath JW. 1999. The finer things in X-ray diffraction data collection. Acta
Crystallogr D Biol Crystallogr. 55:1718–1725.
Qasba PK, Ramakrishnan B, Boeggeman E. 2005. Substrate-induced conformational changes in glycosyltransferases. Trends Biochem Sci. 30:53–62.
Qin BY, Bewley MC, Creamer LK, Baker HM, Baker EN, Jameson GB. 1998.
Structural basis of the Tanford transition of bovine beta-lactoglobulin.
Biochemistry. 37:14014–14023.
Seto NO, Compston CA, Evans SV, Bundle DR, Narang SA, Palcic MM. 1999.
Donor substrate specificity of recombinant human blood group A, B and
hybrid A/B glycosyltransferases expressed in Escherichia coli. Eur J
Biochem. 259:770–775.
Seto NO, Compston CA, Szpacenko A, Palcic MM. 2000. Enzymatic synthesis
of blood group A and B trisaccharide analogues. Carbohydr Res.
324:161–169.
Seto NO, Palcic MM, Compston CA, Li H, Bundle DR, Narang SA. 1997.
Sequential interchange of four amino acids from blood group B to blood
group A glycosyltransferase boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes. J Biol Chem.
272:14133–14138.
Seto NO, Palcic MM, Hindsgaul O, Bundle DR, Narang SA. 1995. Expression
of a recombinant human glycosyltransferase from a synthetic gene and its
utilization for synthesis of the human blood group B trisaccharide. Eur J
Biochem. 234:323–328.
Sharp KA, Honig B. 1990. Electrostatic interactions in macromolecules:
Theory and applications. Annu Rev Biophys Biophys Chem. 19:301–332.
Shoemaker GK, Soya N, Palcic MM, Klassen JS. 2008. Temperature-dependent
cooperativity in donor-acceptor substrate binding to the human blood group
glycosyltransferases. Glycobiology. 18:587–592.
Sinnott ML. 1990. Catalytic mechanisms of enzymatic glycosyl transfer. Chem
Rev. 90:1171–1202.
Sondergaard CR, Olsson MHM, Rostkowski M, Jensen JH. 2011. Improved
treatment of ligands and coupling effects in empirical calculation and rationalization of pK(a) values. J Chem Theory Comput. 7:2284–2295.
Soya N, Shoemaker GK, Palcic MM, Klassen JS. 2009. Comparative study of
substrate and product binding to the human ABO(H) blood group glycosyltransferases. Glycobiology. 19:1224–1234.
Su Y, Yamashita MM, Greasley SE, Mullen CA, Shim JH, Jennings PA,
Benkovic SJ, Wilson IA. 1998. A pH-dependent stabilization of an active
site loop observed from low and high pH crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 A. J Mol Biol.
281:485–499.
Swietnicki W, Petersen R, Gambetti P, Surewicz WK. 1997. pH-dependent stability and conformation of the recombinant human prion protein PrP(90–
231). J Biol Chem. 272:27517–27520.
Thiyagarajan N, Pham TT, Stinson B, Sundriyal A, Tumbale P,
Lizotte-Waniewski M, Brew K, Acharya KR. 2012. Structure of a
metal-independent bacterial glycosyltransferase that catalyzes the synthesis
of histo-blood group A antigen. Sci Rep. 2:940.
245
AR Johal et al.
Unligil UM, Rini JM. 2000. Glycosyltransferase structure and mechanism.
Curr Opin Struct Biol. 10:510–517.
van Stokkum IH, Spoelder HJ, Bloemendal M, van Grondelle R, Groen FC.
1990. Estimation of protein secondary structure and error analysis from circular dichroism spectra. Anal Biochem. 191:110–118.
Watkins WM. 1980. Biochemistry and genetics of the ABO, Lewis, and P
blood group systems. Adv Hum Genet. 10:1–136, 379–385.
Whitmore L, Wallace BA. 2008. Protein secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases. Biopolymers.
89:392–400.
246
Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan
RM, Krissinel EB, Leslie AG, McCoy A, et al. 2011. Overview of the CCP4
suite and current developments. Acta Crystallogr D Biol Crystallogr.
67:235–242.
Wood JL. 1974. pH-controlled hydrogen-bonding. Biochem J. 143:775–777.
Yamamoto F, Clausen H, White T, Marken J, Hakomori S. 1990. Molecular
genetic basis of the histo-blood group ABO system. Nature. 345:229–233.
Yamamoto F, Hakomori S. 1990. Sugar-nucleotide donor specificity of histoblood group A and B transferases is based on amino acid substitutions.
J Biol Chem. 265:19257–19262.