* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download pH-induced conformational changes in human ABO(H) blood group
Survey
Document related concepts
Interactome wikipedia , lookup
Two-hybrid screening wikipedia , lookup
NADH:ubiquinone oxidoreductase (H+-translocating) wikipedia , lookup
Enzyme inhibitor wikipedia , lookup
Oxidative phosphorylation wikipedia , lookup
Biosynthesis wikipedia , lookup
Protein–protein interaction wikipedia , lookup
X-ray crystallography wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Biochemistry wikipedia , lookup
Proteolysis wikipedia , lookup
Homology modeling wikipedia , lookup
Catalytic triad wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Anthrax toxin wikipedia , lookup
Transcript
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.