Monoclonal antibody 7H2.2 binds the C-terminus of the cancer-oocyte antigen SAS1B through the hydrophilic face of a conserved amphipathic helix corresponding to one of only two regions predicted to be ordered.

The structure of the antigen-binding fragment (Fab) of mouse monoclonal antibody 7H2.2 in complex with a 15-residue fragment from the metalloproteinase sperm acrosomal SLLP1 binding protein (SAS1B), which is a molecular and cellular candidate for both cancer therapy and female contraception, has been determined at 2.75 Šresolution by single-crystal X-ray diffraction. Although the crystallization conditions contained the final 148 C-terminal residues of SAS1B, the Fab was observed to crystallize in complex with a 15-residue fragment corresponding to one of only two elements of secondary structure that are predicted to be ordered within the C-terminal region of SAS1B. The antigen forms an amphipathic α-helix that binds the 7H2.2 combining site via hydrophilic residues in an epitope that spans the length of the antigen α-helix, with only two CH-π interactions observed along the edge of the interface between the antibody and antigen. Interestingly, the paratope contains two residues mutated away from the germline (YL32F and YH58R), as well as a ProH96-ThrH97-AspH98-AspH99 insertion within heavy chain CDR3. The intact 7H2.2 antibody exhibits high affinity for the SAS1B antigen, with 1:1 binding and nanomolar affinity for both the SAS1B C-terminal construct used for crystallization (3.38 ± 0.59 nM) and a 15-amino-acid synthetic peptide construct corresponding to the helical antigen observed within the crystal structure (1.60 ± 0.31 nM). The SAS1B-antibody structure provides the first structural insight into any portion of the subdomain architecture of the C-terminal region of the novel cancer-oocyte tumor surface neoantigen SAS1B and provides a basis for the targeted use of SAS1B.

Antigen binding by conformational selection in near-germline antibodies.

Conformational flexibility in antibody-combining sites has been hypothesized to facilitate polyspecificity toward multiple unique epitopes and enable the limited germline repertoire to match an overwhelming diversity of potential antigens; however, elucidating the mechanisms of antigen recognition by flexible antibodies has been understandably challenging. Here, multiple liganded and unliganded crystal structures of the near-germline anticarbohydrate antibodies S25-2 and S25-39 are reported, which reveal an unprecedented diversity of complementarity-determining region H3 conformations in apparent equilibrium. These structures demonstrate that at least some germline or near-germline antibodies are flexible entities sensitive to their chemical environments, with conformational selection available as an evolved mechanism that preserves the inherited ability to recognize common pathogens while remaining adaptable to new threats.

The S-layer homology domains of Paenibacillus alvei surface protein SpaA bind to cell wall polysaccharide through the terminal monosaccharide residue.

Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaASLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaASLH accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.

Subtle Changes in the Combining Site of the Chlamydiaceae-Specific mAb S25-23 Increase the Antibody-Carbohydrate Binding Affinity by an Order of Magnitude.

Murine antibodies S25-23, S25-26, and S25-5 derive from a common germ-line origin, and all bind the Chlamydiaceae family-specific epitope αKdo(2→8)αKdo(2→4)αKdo (where Kdo is 3-deoxy-α-d- manno-oct-2-ulosonic acid) with high affinity and specificity. These antibodies recognize the entire trisaccharide antigen in a linkage-dependent manner via a groove composed largely of germ-line residues. Despite sharing identical heavy and light chain genes, S25-23 binds the family-specific epitope with nanomolar affinity, which is an order of magnitude higher than that of S25-26, while S25-5 displays an affinity between those of S25-23 and S25-26. We determined the high-resolution crystal structures of S25-23 and S25-5 antigen binding fragments in complex with a pentasaccharide derived from the LPS of Chlamydia and measured the affinity of S25-5 for chlamydial LPS antigens using isothermal titration microcalorimetry. The 1.75 Å resolution structure of S25-23 shows how subtle conservative mutations Arg(L)-27E to lysine and Ser(H)-56 to threonine lead to an order of magnitude increase in affinity. Importantly, comparison between previous S25-26 structures and the 1.99 and 2.05 Å resolution liganded and unliganded structures of S25-5, respectively, shows how a Ser(L)-27E mutation results in an intermediate affinity due to the reduced enthalpic penalty associated with complex formation that would otherwise be required for arginine in this position. This strategy allows for subtle adjustments in the combining site via affinity maturation that have dramatic consequences for the affinity of an antibody for its antigen.

Structural basis of cell wall anchoring by SLH domains in Paenibacillus alvei.

Self-assembling protein surface (S-) layers are common cell envelope structures of prokaryotes and have critical roles from structural maintenance to virulence. S-layers of Gram-positive bacteria are often attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Here we present an in-depth characterization of this interaction, with co-crystal structures of the three consecutive SLH domains from the Paenibacillus alvei S-layer protein SpaA with defined SCWP ligands. The most highly conserved SLH domain residue SLH-Gly29 is shown to enable a peptide backbone flip essential for SCWP binding in both biophysical and cellular experiments. Furthermore, we find that a significant domain movement mediates binding by two different sites in the SLH domain trimer, which may allow anchoring readjustment to relieve S-layer strain caused by cell growth and division.

Conserved residues Arg188 and Asp302 are critical for active site organization and catalysis in human ABO(H) blood group A and B glycosyltransferases.

Homologous glycosyltransferases GTA and GTB perform the final step in human ABO(H) blood group A and B antigen synthesis by transferring the sugar moiety from donor UDP-GalNAc/UDP-Gal to the terminal H antigen disaccharide acceptor. Like other GT-A fold family 6 glycosyltransferases, GTA and GTB undergo major conformational changes in two mobile regions, the C-terminal tail and internal loop, to achieve the closed, catalytic state. These changes are known to establish a salt bridge network among conserved active site residues Arg188, Asp211 and Asp302, which move to accommodate a series of discrete donor conformations while promoting loop ordering and formation of the closed enzyme state. However, the individual significance of these residues in linking these processes remains unclear. Here, we report the kinetics and high-resolution structures of GTA/GTB mutants of residues 188 and 302. The structural data support a conserved salt bridge network critical to mobile polypeptide loop organization and stabilization of the catalytically competent donor conformation. Consistent with the X-ray crystal structures, the kinetic data suggest that disruption of this salt bridge network has a destabilizing effect on the transition state, emphasizing the importance of Arg188 and Asp302 in the glycosyltransfer reaction mechanism. The salt bridge network observed in GTA/GTB structures during substrate binding appears to be conserved not only among other Carbohydrate Active EnZyme family 6 glycosyltransferases but also within both retaining and inverting GT-A fold glycosyltransferases. Our findings augment recently published crystal structures, which have identified a correlation between donor substrate conformational changes and mobile loop ordering.

Polyspecificity of Anti-lipid A Antibodies and Its Relevance to the Development of Autoimmunity.

The process of natural selection favours germ-line gene segments that encode CDRs that have the ability to recognize a range of structurally related antigens. This presents an immunological advantage to the host, as it can confer protection against a common pathogen and still cope with new or changing antigens. Cross-reactive and polyspecific antibodies also play a central role in autoimmune responses, and a link has been shown to exist between auto-reactive B cells and certain bacterial infections. Bacterial DNA, lipids, and carbohydrates have been implicated in the progression of autoimmune diseases such as systemic lupus erythematosus. As well, reports of anti-lipid A antibody polyspecificity towards single-stranded DNA together with the observed sequence homology amongst isolated auto- and anti-lipid A antibodies has prompted further study of this phenomenon. Though the lipid A epitope appears cryptic during Gram-negative bacterial infection, there have been several reported instances of lipid A-specific antibodies isolated from human sera, some of which have exhibited polyspecificity for single stranded DNA. In such cases, the breakdown of negative selection through polyspecificity can have the unfortunate consequence of autoimmune disease. This review summarizes current knowledge regarding such antibodies and emphasizes the features of S1-15, A6, and S55-5, anti-lipid A antibodies whose structures were recently determined by X-ray crystallography.

High-resolution crystal structures and STD NMR mapping of human ABO(H) blood group glycosyltransferases in complex with trisaccharide reaction products suggest a molecular basis for product release.

The human ABO(H) blood group A- and B-synthesizing glycosyltransferases GTA and GTB have been structurally characterized to high resolution in complex with their respective trisaccharide antigen products. These findings are particularly timely and relevant given the dearth of glycosyltransferase structures collected in complex with their saccharide reaction products. GTA and GTB utilize the same acceptor substrates, oligosaccharides terminating with α-l-Fucp-(1→2)-ß-d-Galp-OR (where R is a glycolipid or glycoprotein), but use distinct UDP donor sugars, UDP-N-acetylgalactosamine and UDP-galactose, to generate the blood group A (α-l-Fucp-(1→2)[α-d-GalNAcp-(1→3)]-ß-d-Galp-OR) and blood group B (α-l-Fucp-(1→2)[α-d-Galp-(1→3)]-ß-d-Galp-OR) determinant structures, respectively. Structures of GTA and GTB in complex with their respective trisaccharide products reveal a conflict between the transferred sugar monosaccharide and the ß-phosphate of the UDP donor. Mapping of the binding epitopes by saturation transfer difference NMR measurements yielded data consistent with the X-ray structural results. Taken together these data suggest a mechanism of product release where monosaccharide transfer to the H-antigen acceptor induces active site disorder and ejection of the UDP leaving group prior to trisaccharide egress.

Glycosyltransfer in mutants of putative catalytic residue Glu303 of the human ABO(H) A and B blood group glycosyltransferases GTA and GTB proceeds through a labile active site.

The homologous glycosyltransferases α-1,3-N-acetylgalactosaminyltransferase (GTA) and α-1,3-galactosyltransferase (GTB) carry out the final synthetic step of the closely related human ABO(H) blood group A and B antigens. The catalytic mechanism of these model retaining enzymes remains under debate, where Glu303 has been suggested to act as a putative nucleophile in a double displacement mechanism, a local dipole stabilizing the intermediate in an orthogonal associative mechanism or a general base to stabilize the reactive oxocarbenium ion-like intermediate in an SNi-like mechanism. Kinetic analysis of GTA and GTB point mutants E303C, E303D, E303Q and E303A shows that despite the enzymes having nearly identical sequences, the corresponding mutants of GTA/GTB have up to a 13-fold difference in their residual activities relative to wild type. High-resolution single crystal X-ray diffraction studies reveal, surprisingly, that the mutated Cys, Asp and Gln functional groups are no more than 0.8 Å further from the anomeric carbon of donor substrate compared to wild type. However, complicating the analysis is the observation that Glu303 itself plays a critical role in maintaining the stability of a strained "double-turn" in the active site through several hydrogen bonds, and any mutation other than E303Q leads to significantly higher thermal motion or even disorder in the substrate recognition pockets. Thus, there is a remarkable juxtaposition of the mutants E303C and E303D, which retain significant activity despite disrupted active site architecture, with GTB/E303Q, which maintains active site architecture but exhibits zero activity. These findings indicate that nucleophilicity at position 303 is more catalytically valuable than active site stability and highlight the mechanistic elasticity of these enzymes.

Molecular Basis for Recognition of the Cancer Glycobiomarker, LacdiNAc (GalNAc[ß1→4]GlcNAc), by Wisteria floribunda Agglutinin.

Aberrant glycosylation and the overexpression of specific carbohydrate epitopes is a hallmark of many cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy and diagnostics. Wisteria floribunda agglutinin (WFA) is a legume lectin that recognizes terminal N-acetylgalactosaminides with high affinity. WFA preferentially binds the disaccharide LacdiNAc (ß-d-GalNAc-[1→4]-d-GlcNAc), which is associated with tumor malignancy in leukemia, prostate, pancreatic, ovarian, and liver cancers and has shown promise in cancer glycobiomarker detection. The mechanism of specificity for WFA recognition of LacdiNAc is not fully understood. To address this problem, we have determined affinities and structure of WFA in complex with GalNAc and LacdiNAc. Affinities toward Gal, GalNAc, and LacdiNAc were measured via surface plasmon resonance, yielding KD values of 4.67 × 10-4 m, 9.24 × 10-5 m, and 5.45 × 10-6 m, respectively. Structures of WFA in complex with LacdiNAc and GalNAc have been determined to 1.80-2.32 Å resolution. These high resolution structures revealed a hydrophobic groove complementary to the GalNAc and, to a minor extent, to the back-face of the GlcNAc sugar ring. Remarkably, the contribution of this small hydrophobic surface significantly increases the observed affinity for LacdiNAc over GalNAc. Tandem MS sequencing confirmed the presence of two isolectin forms in commercially available WFA differing only in the identities of two amino acids. Finally, the WFA carbohydrate binding site is similar to a homologous lectin isolated from Vatairea macrocarpa in complex with GalNAc, which, unlike WFA, binds not only αGalNAc but also terminal Ser/Thr O-linked αGalNAc (Tn antigen).

The Combining Sites of Anti-lipid A Antibodies Reveal a Widely Utilized Motif Specific for Negatively Charged Groups.

Lipopolysaccharide dispersed in the blood by Gram-negative bacteria can be a potent inducer of septic shock. One research focus has been based on antibody sequestration of lipid A (the endotoxic principle of LPS); however, none have been successfully developed into a clinical treatment. Comparison of a panel of anti-lipid A antibodies reveals highly specific antibodies produced through distinct germ line precursors. The structures of antigen-binding fragments for two homologous mAbs specific for lipid A, S55-3 and S55-5, have been determined both in complex with lipid A disaccharide backbone and unliganded. These high resolution structures reveal a conserved positively charged pocket formed within the complementarity determining region H2 loops that binds the terminal phosphates of lipid A. Significantly, this motif occurs in unrelated antibodies where it mediates binding to negatively charged moieties through a range of epitopes, including phosphorylated peptides used in diagnostics and therapeutics. S55-3 and S55-5 have combining sites distinct from anti-lipid A antibodies previously described (as a result of their separate germ line origin), which are nevertheless complementary both in shape and charge to the antigen. S55-3 and S55-5 display similar avidity toward lipid A despite possessing a number of different amino acid residues in their combining sites. Binding of lipid A occurs independent of the acyl chains, although the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains their inability to recognize LPS. Despite their lack of therapeutic potential, the observed motif may have significant immunological implications as a tool for engineering recombinant antibodies.

Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner†.

The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ∼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition.

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner.

Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the "tucked under" conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be "isosteric" with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB.

Antibody recognition of carbohydrate epitopes†.

Carbohydrate antigens are valuable as components of vaccines for bacterial infectious agents and human immunodeficiency virus (HIV), and for generating immunotherapeutics against cancer. The crystal structures of anti-carbohydrate antibodies in complex with antigen reveal the key features of antigen recognition and provide information that can guide the design of vaccines, particularly synthetic ones. This review summarizes structural features of anti-carbohydrate antibodies to over 20 antigens, based on six categories of glyco-antigen: (i) the glycan shield of HIV glycoproteins; (ii) tumor epitopes; (iii) glycolipids and blood group A antigen; (iv) internal epitopes of bacterial lipopolysaccharides; (v) terminal epitopes on polysaccharides and oligosaccharides, including a group of antibodies to Kdo-containing Chlamydia epitopes; and (vi) linear homopolysaccharides.

Structural Basis for Antibody Recognition of Lipid A: INSIGHTS TO POLYSPECIFICITY TOWARD SINGLE-STRANDED DNA.

Septic shock is a leading cause of death, and it results from an inflammatory cascade triggered by the presence of microbial products in the blood. Certain LPS from Gram-negative bacteria are very potent inducers and are responsible for a high percentage of septic shock cases. Despite decades of research, mAbs specific for lipid A (the endotoxic principle of LPS) have not been successfully developed into a clinical treatment for sepsis. To understand the molecular basis for the observed inability to translate in vitro specificity for lipid A into clinical potential, the structures of antigen-binding fragments of mAbs S1-15 and A6 have been determined both in complex with lipid A carbohydrate backbone and in the unliganded form. The two antibodies have separate germ line origins that generate two markedly different combining-site pockets that are complementary both in shape and charge to the antigen. mAb A6 binds lipid A through both variable light and heavy chain residues, whereas S1-15 utilizes exclusively the variable heavy chain. Both antibodies bind lipid A such that the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains the lack of LPS recognition. Longstanding reports of polyspecificity of anti-lipid A antibodies toward single-stranded DNA combined with observed homology of S1-15 and A6 and the reports of several single-stranded DNA-specific mAbs prompted the determination of the structure of S1-15 in complex with single-stranded DNA fragments, which may provide clues about the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases.

Groove-type recognition of chlamydiaceae-specific lipopolysaccharide antigen by a family of antibodies possessing an unusual variable heavy chain N-linked glycan.

The structure of the antigen binding fragment of mAb S25-26, determined to 1.95 Å resolution in complex with the Chlamydiaceae family-specific trisaccharide antigen Kdo(2→8)Kdo(2→4)Kdo (Kdo = 3-deoxy-α-d-manno-oct-2-ulopyranosonic acid), displays a germ-line-coded paratope that differs significantly from previously characterized Chlamydiaceae-specific mAbs despite being raised against the identical immunogen. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germ-line residues to recognize the entire trisaccharide antigen and so confer strict specificity. Interest in S25-23 was sparked by its rare high µm affinity and strict specificity for the family-specific trisaccharide antigen; however, only the related antibody S25-26 proved amenable to crystallization. The structures of three unliganded forms of S25-26 have a labile complementary-determining region H3 adjacent to significant glycosylation of the variable heavy chain on asparagine 85 in Framework Region 3. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the few reported structures of glycosylated mAbs containing these epitopes is the therapeutic antibody Cetuximab; however, unlike Cetuximab, one of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three-dimensional structure of an αGal-containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as it has been implicated in allergic response in patients receiving therapeutic antibodies.

Structural basis for selective cross-reactivity in a bactericidal antibody against inner core lipooligosaccharide from Neisseria meningitidis.

The structure of a antigen-binding fragment (Fab) from the bactericidal monoclonal antibody LPT3-1 specific to lipooligosaccharide (LOS) inner cores from Neisseria meningitidis has been solved in complex with an eight-sugar inner core fragment NmL3 galE lpt3 KOH to 2.69 Å resolution. The epitope is centered about an inner core N-acetylglucosamine residue unique to N. meningitidis and does not include the lipid A moiety, which is disordered in the structure, but is positioned to allow the binding of free and membrane-anchored full-length LOS. All the amino acid residues that contact antigen are of germline origin but, remarkably, two consecutive somatic mutations of serine to glycine in the heavy chain at residues 52 and 52a are positioned to deprive the antibody of advantageous interactions and so weaken binding. However, these mutations are key to allowing selective cross-reactivity with the HepII-3-PEtn inner core variant expressed by 70% of strains. Neisseria meningitidis is a leading cause of disease in the developed world and is especially dangerous to children, who lack the necessary protective antibodies. The structure of Fab LPT3-1 in complex with LOS provides insight into the antibody's selective ability to recognize multiple clinically relevant variations of the LOS inner core from N. meningitidis.

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.

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.

Geometric attributes of retaining glycosyltransferase enzymes favor an orthogonal mechanism.

Retaining glycosyltransferase enzymes retain the stereochemistry of the donor glycosidic linkage after transfer to an acceptor molecule. The mechanism these enzymes utilize to achieve retention of the anomeric stereochemistry has been a matter of much debate. Re-analysis of previously released structural data from retaining and inverting glycosyltransferases allows competing mechanistic proposals to be evaluated. The binding of metal-nucleotide-sugars between inverting and retaining enzymes is conformationally unique and requires the donor substrate to occupy two different orientations in the two types of glycosyltransferases. The available structures of retaining glycosyltransferases lack appropriately positioned enzymatic dipolar residues to initiate or stabilize the intermediates of a dissociative mechanism. Further, available structures show that the acceptor nucleophile and anomeric carbon of the donor sugar are in close proximity. Structural features support orthogonal (front-side) attack from a position lying ≤ 90° from the C1-O phosphate bond for retaining enzymes. These structural conclusions are consistent with the geometric conclusions of recent kinetic and computational studies.