RESUMEN
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.
Asunto(s)
Anticuerpos , Regiones Determinantes de Complementariedad , Anticuerpos/química , Sitios de Unión de Anticuerpos , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Cristalografía por Rayos X , Células Germinativas , Conformación Molecular , Conformación ProteicaRESUMEN
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.
Asunto(s)
Pared Celular/química , Paenibacillus/citología , Peptidoglicano/química , Secuencias de Aminoácidos , Bacillus anthracis , Proliferación Celular , Dicroismo Circular , Cristalización , Ligandos , Mutagénesis , Mutagénesis Sitio-Dirigida , Unión Proteica , Dominios Proteicos , Proteínas Recombinantes/químicaRESUMEN
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.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/genética , Antígenos de Grupos Sanguíneos/genética , Galactosiltransferasas/genética , Sistema del Grupo Sanguíneo ABO/química , Sistema del Grupo Sanguíneo ABO/inmunología , Secuencia de Aminoácidos/genética , Antígenos de Grupos Sanguíneos/química , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Galactosiltransferasas/química , Humanos , Enlace de Hidrógeno , Cinética , Mutación , Mutación Puntual , Especificidad por SustratoRESUMEN
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 â 2)αMan(1 â 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 â¼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition.
Asunto(s)
Manosafosfatos/química , Anticuerpos de Cadena Única/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Ratones , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Anticuerpos de Cadena Única/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antivirales/inmunología , Carbohidratos/inmunología , Epítopos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/química , Anticuerpos Antineoplásicos/química , Anticuerpos Antivirales/química , Carbohidratos/química , Epítopos/química , Epítopos/genética , Humanos , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/química , Glicosiltransferasas/química , Secuencia de Aminoácidos , Dominio Catalítico , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Conformación Proteica , Electricidad EstáticaRESUMEN
The crystal structures of the antigen-binding fragment of the murine monoclonal antibody (mAb) S25-39 in the presence of several antigens representing chlamydial lipopolysaccharide (LPS) epitopes based on the bacterial sugar 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) have been determined at resolutions from 2.4 to 1.8 Å. The antigen-binding site of this antibody differs from the well-characterized antibody S25-2 by a single mutation away from the germline of asparagine H53 to lysine, yet this one mutation results in a significant increase in avidity across a range of antigens. A comparison of the two antibody structures reveals that the mutated Lys H53 forms additional hydrogen bonds and/or charged-residue interactions with the second Kdo residue of every antigen having two or more carbohydrate residues. Significantly, the NH53K mutation results from a single nucleotide substitution in the germline sequence common among a panel of antibodies raised against glycoconjugates containing carbohydrate epitopes of chlamydial LPS. Like S25-2, S25-39 displays significant induced fit of complementarity determining region (CDR) H3 upon antigen binding, with the unliganded structure possessing a conformation distinct from those reported earlier for S25-2. The four different observed conformations for CDR H3 suggest that this CDR has evolved to exploit the recognition potential of a flexible loop while minimizing the associated entropic penalties of binding by adopting a limited number of ordered conformations in the unliganded state. These observations reveal strategies evolved to balance adaptability and specificity in the germline antibody response to carbohydrate antigens.
Asunto(s)
Sitios de Unión de Anticuerpos/inmunología , Chlamydia/inmunología , Lipopolisacáridos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Antígenos Bacterianos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Epítopos/genética , Epítopos/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Cadenas Pesadas de Inmunoglobulina/genética , RatonesRESUMEN
In order to explore the structural basis for adaptability in near germline monoclonal antibodies (mAb), we have examined the specificity of the promiscuous mAb S67-27 to both naturally derived carbohydrate antigens and a variety of synthetic nonnatural antigens based on the bacterial lipopolysaccharide component 3-deoxy-alpha-D-manno-oct-2-ulosonic acid (Kdo). One such analog, a 7-O-methyl (7-O-Me) Kdo disaccharide, was found to bind to the antibody with at least 30-fold higher affinity than any other antigen tested. The structure of S67-27 in complex with this analog and three other naturally occurring Kdo antigens revealed that the enhanced affinity of the mAb for the synthetic analog was accomplished by the strategic positioning of CDR H3 away from a conserved Kdo binding pocket that allowed the formation of new antibody-antigen contacts. Furthermore, the comparison of this structure with the structures of related mAbs revealed how the position and structure of CDR H3 influence the specificity or promiscuity of near-germline carbohydrate-recognizing antibodies by altering the architecture of the combining site.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Lipopolisacáridos/inmunología , Azúcares Ácidos/inmunología , Animales , Reacciones Antígeno-Anticuerpo , Antígenos Bacterianos/química , Lipopolisacáridos/síntesis química , Lipopolisacáridos/química , Ratones , Ratones Endogámicos BALB C , Azúcares Ácidos/síntesis química , Azúcares Ácidos/químicaRESUMEN
A difference of seven conservative amino-acid substitutions between two single-chain antibodies (scFvs) specific for chlamydial lipopolysaccharide does not significantly affect their molecular structures or packing contacts, but dramatically affects their crystallization. The structure of the variable domain (Fv) of SAG173-04 was solved to 1.86 A resolution and an R(cryst) of 18.9% in space group P2(1)2(1)2(1). Crystals of the homologous SAG506-01 diffracted to 1.95 A resolution and appeared at first to have Patterson symmetry I4/m or P4/mmm; however, no solution could be found in space groups belonging to the former and refinement in the only solution corresponding to the latter (in space group P4(3)2(1)2) stalled at R(free) = 30.0%. Detailed examination of the diffraction data revealed that the crystal was likely to be twinned and that the correct space group was P2(1)2(1)2(1). Both translational pseudo-symmetry and pseudo-merohedral twinning were observed in one crystal of SAG506-01 and pseudo-merohedral twinning was observed for a second crystal. The final R factor for SAG506-01 after refinement in P2(1)2(1)2(1) was 20.5%.