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The crystal structure of the murine Fab S-20-4 from a protective anti-cholera Ab specific for the lipopolysaccharide Ag of the Ogawa serotype has been determined in its unliganded form and in complex with synthetic fragments of the Ogawa O-specific polysaccharide (O-SP). The upstream terminal O-SP monosaccharide is shown to be the primary antigenic determinant. Additional perosamine residues protrude outwards from the Ab surface and contribute only marginally to the binding affinity and specificity. A complementary water-excluding hydrophobic interface and five Ab-Ag hydrogen bonds are crucial for carbohydrate recognition. The structure reported here explains the serotype specificity of anti-Ogawa Abs and provides a rational basis toward the development of a synthetic carbohydrate-based anti-cholera vaccine.

Studies have been performed with platelets using C1 haemolytic assays and platelet aggregation induced by anti-C1q, anti-C1s and aggregated IgG in the presence of C1 subcomponents C1q, C1r and C1s. C1q was removed by EDTA or modified by collagenase from human platelets while after the same treatment C1s remained bound to the platelets. EDTA treated platelets were no longer aggregated by aggregated IgG. The addition of C1q restored the reactivity of the platelets to aggregated IgG while the addition of C1r or C1s was without effect. Furthermore, the addition of C1r or C1s to C1q inhibited the action of C1q in platelet aggregation induced by IgG. The possible association between the different C1 subcomponents and human platelets is discussed. [ABSTRACT FROM AUTHOR]

A longstanding debate concerns the function of carinated elements in both, the Levantine, and European Aurignacian. The present study aims to contribute to this topic with the evaluation of the carinated assemblage from layer D in Hayonim Cave, Western Galilee, Israel, one of the type sites of the Levantine Aurignacian. An operational chain reconstruction with an attribute analysis is paired with a typological approach to the preparation and maintenance products based on artefacts defined as West European Aurignacian. The results of this study are investigated with multivariate statistics offering a methodological contribution. The data is subjected to a transformation into a distance matrix using the Gower distance and tested with the adonis-algorithm for significance. The results clearly indicate that the carinated items in Hayonim Cave did fully or partially function as cores. They are accompanied by diagnostic preparation- and- maintenance products known from the literature e.g. Thèmes bladelets. The statistical analysis indicates only a minor correlation with stratigraphy yet supports the techno-typological criteria applied for defining artefact categories (cores, debitage, tools), as well as the proposed differentiation of carinated 'core' types. The non-carinated cores in Hayonim Cave are characterised by a high variability in typology and reduction concepts. A curious similarity to the Levallois-concept is observed on some of the flake cores. It is therefore suggested that the frequent recycling of Middle Palaeolithic artefacts in the Levantine Aurignacian might have given the Aurignacian flint-knappers the opportunity to study the Levallois concept and apply an approximation of it in their own core reduction strategies. The notion that Palaeolithic flint-knappers actively observed former technological systems through the discarded artefacts directly opens up a new trajectory for the understanding of lithic reduction concept permanence. The conceptual diversification and variability in Hayonim Cave D indicate a highly dynamic period in the Levantine Upper Palaeolithic which increased the adaptive potential and promoted a rapid cultural change., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Parow-Souchon, Belfer-Cohen. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Our field data from the Upper Palaeolithic site of Al-Ansab 1 (Jordan) and from a pollen sequence in the Dead Sea elucidate the role that changing Steppe landscapes played in facilitating anatomically modern human populations to enter a major expansion and consolidation phase, known as the "Early Ahmarian", several millennia subsequent to their initial Marine Isotope Stage 4/3 migration from Africa, into the Middle East. The Early Ahmarian techno-cultural unit covers a time range between 45 ka-37 ka BP. With so far more than 50 sites found, the Early Ahmarian is the first fully Upper Palaeolithic techno-cultural unit exclusively and undisputedly related to anatomically modern human populations. In order to better understand the potentially attractive features of the Early Ahmarian environmental context that supported its persistence for over 8,000 years, we carried out a decennial research program in Jordan and in the Dead Sea. This included (1) a geoscientific and archaeological survey program in the Wadi Sabra (Jordan) with a particular focus on excavations at the Early Ahmarian site of Al-Ansab 1 alongside the detailed analysis of Quaternary sediments from the same area and (2) palaeobotanical research based on Quaternary lake deposits from the Dead Sea. Our pollen data from the Dead Sea indicate slow, low frequency vegetational variation with expanding Artemisia steppe, from 60 to 20 ka BP (MIS 3-2). Here, we see a reciprocal assimilation of southern and northern Levantine vegetation zones thereby enhancing a long-lasting south-to-north steppe corridor. The same integration process accelerated about 40 ka ago, when forested areas retreated in the Lebanese Mountains. The process then extended to encompass an area from Southern Lebanon to the Sinai Peninsula. We argue that, at the same time, the carriers of the Early Ahmarian techno-cultural unit extended their habitat from their original Mediterranean biome (in the North) to the Saharo-Arabian biome (to the South). Our excavation of Al-Ansab 1, a campsite at the eastern margins of the Early Ahmarian settlement area, indicates far reaching annual movements of small, highly mobile hunter-gatherer groups. We assume a low degree of settlement complexity, still allowing for habitat extension of the Early Ahmarian into the margins of the Levantine corridor. Due to our radiometric dates, our combined archaeological and environmental record sheds light on an evolved phase of the Early Ahmarian, around 38 ka ago, rather than the starting phase of this techno-cultural unit. Possible application of our model to the starting phase of the Early Ahmarian remains an aspect of future research., Competing Interests: The authors have declared that no competing interests exist.

The peroxiredoxin AhpC from Mycobacterium tuberculosis (MtAhpC) is the foremost element of a NADH-dependent peroxidase and peroxynitrite reductase system, where it directly reduces peroxides and peroxynitrite and is in turn reduced by AhpD and other proteins. Overexpression of MtAhpC in isoniazid-resistant strains of M. tuberculosis harboring mutations in the catalase/peroxidase katG gene provides antioxidant protection and may substitute for the lost enzyme activities. We report here the crystal structure of oxidized MtAhpC trapped in an intermediate oligomeric state of its catalytic cycle. The overall structure folds into a ring-shaped hexamer of dimers instead of the usual pentamer of dimers observed in other reduced peroxiredoxins. Although the general structure of the functional dimer is similar to that of other 2-Cys peroxiredoxins, the alpha-helix containing the peroxidatic cysteine Cys61 undergoes a unique rigid-body movement to allow the formation of the disulfide bridge with the resolving cysteine Cys174. This conformational rearrangement creates a large internal cavity enclosing the active site, which might be exploited for the design of inhibitors that could block the catalytic cycle. Structural and mutagenesis evidence points to a model for the electron transfer pathway in MtAhpC that accounts for the unusual involvement of three cysteine residues in catalysis and suggests a mechanism by which MtAhpC can specifically interact with different redox partners.

Cellobiohydrolase CelS plays an important role in the cellulosome, an active cellulase system produced by the thermophilic anaerobe Clostridium thermocellum. The structures of the catalytic domain of CelS in complex with substrate (cellohexaose) and product (cellobiose) were determined at 2.5 and 2.4 A resolution, respectively. The protein folds into an (alpha/alpha)(6) barrel with a tunnel-shaped substrate-binding region. The conformation of the loops defining the tunnel is intrinsically stable in the absence of substrate, suggesting a model to account for the processive mode of action of family 48 cellobiohydrolases. Structural comparisons with other (alpha/alpha)(6) barrel glycosidases indicate that CelS and endoglucanase CelA, a sequence-unrelated family 8 glycosidase with a groove-shaped substrate-binding region, use the same catalytic machinery to hydrolyze the glycosidic linkage, despite a low sequence similarity and a different endo/exo mode of action. A remarkable feature of the mechanism is the absence, from CelS, of a carboxylic group acting as the base catalyst. The nearly identical arrangement of substrate and functionally important residues in the two active sites strongly suggests an evolutionary relationship between the cellobiohydrolase and endoglucanase families, which can therefore be classified into a new clan of glycoside hydrolases., ((c) 2002 Elsevier Science Ltd.)

The crystal structure of Clostridium thermocellum endoglucanase CelA in complex with cellopentaose has been determined at 0.94 A resolution. The oligosaccharide occupies six D-glucosyl-binding subsites, three on either side of the scissile glycosidic linkage. The substrate and product of the reaction occupy different positions at the reducing end of the cleft, where an extended array of hydrogen-bonding interactions with water molecules fosters the departure of the leaving group. Severe torsional strain upon the bound substrate forces a distorted boat(2,5) B conformation for the glucosyl residue bound at subsite -1, which facilitates the formation of an oxocarbenium ion intermediate and might favor the breakage of the sugar ring concomitant with catalysis., (Copyright 2002 Elsevier Science Ltd.)

A series of mutants bearing single amino acid substitutions often encountered in the catalase/peroxidase, KatG, from isoniazid-resistant isolates of Mycobacterium tuberculosis has been produced by site-directed mutagenesis. The resultant enzymes were overexpressed, purified and characterized. Replacing Cys-20 by Ser abolished disulphide-bridge formation, but did not affect either dimerization of the enzyme or catalysis. The substitution of Thr-275, which is probably involved in electron transfer from the haem, by proline resulted in a highly unstable enzyme with insignificant enzyme activities. The most commonly occurring substitution in drug-resistant clinical isolates is the replacement of Ser-315 by Thr; this lowered catalase and peroxidase activities by 50% and caused a significant decrease in the KatG-mediated inhibition of the activity of the NADH-dependent enoyl-[acyl-carrier protein] reductase, InhA, in vitro. The ability of this enzyme to produce free radicals from isoniazid was severely impaired, as judged by its loss of NitroBlue Tetrazolium reduction activity. Replacement of Leu-587 by Pro resulted in marked instability of KatG, indicating that the C-terminal domain is also important for structural and functional integrity.

The catalytic domain of cellulase CelA, a family 8 glycohydrolase from C. thermocellum, has been crystallized in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 50.12 A, b = 63.52 A, c = 104.97 A. The diffraction pattern extends beyond 1.5 A resolution.

The structures of the Glu140-->Gln mutant of the Clostridium thermocellum endoglucanase CelC in unliganded form (CelC(E140Q)) and in complex with cellohexaose (CelC(E140Q)-Gl(C6)) have been refined to crystallographic R-factors of 19.4% at 1.9 A and 17.8% at 2.3 A resolution, respectively. The structure of CelC(E140Q)-Gl(C6) complex shows two D-glucosyl residues bound to the non-reducing end of the substrate-binding cleft. Comparison of the unliganded and complexes structures reveals conformational changes due to substrate binding, including a significant reorientation of the loop 138-141 which carries the general acid/base catalyst Glu140 in wild-type CelC. Endoglucanase CelC, a family 5 glycohydrolase, exhibits a (beta/alpha)8-fold with an additional subdomain of 54 amino acids inserted between beta-strand 6 and alpha-helix 6. Seven amino acid residues (Arg46, His90, Asn139, Glu140, His198, Tyr200, and Glu280) located close to the catalytic reaction center are strictly conserved in family 5 cellulases. Only three of these residues (His90, Gln140 and Glu280) make direct contacts with the substrate, but all participate in a network of hydrogen bonds which contribute to the stability of the active site architecture and may influence the protonation state of the two catalytic residues. Residue Trp313, which interacts with the nucleophile Glu280 and is within hydrogen bonding distance of the substrate, is involved in a non-proline cis-peptide bond. An aromatic residue occurs at an equivalent position in many other (beta/alpha)8-barrel glycosidases; the presence of a cis-peptide bond at this position in the structures of family 1 beta-glucosidases, family 2 beta-galactosidases, family 5 cellulases, family 17 beta-glucanases, and family 18 chitinases provides further evidence of an evolutionary relationship between glycosyl hydrolases with a (beta/alpha)8- architecture.

Background: Cellulases, which catalyze the hydrolysis of glycosidic bonds in cellulose, can be classified into several different protein families. Endoglucanase CelA is a member of glycosyl hydrolase family 8, a family for which no structural information was previously available., Results: The crystal structure of CelA was determined by multiple isomorphous replacement and refined to 1.65 A resolution. The protein folds into a regular (alpha/alpha)6 barrel formed by six inner and six outer alpha helices. Cello-oligosaccharides bind to an acidic cleft containing at least five D-glucosyl-binding subsites (A-E) such that the scissile glycosidic linkage lies between subsites C and D. The strictly conserved residue Glu95, which occupies the center of the substrate-binding cleft and is hydrogen bonded to the glycosidic oxygen, has been assigned the catalytic role of proton donor., Conclusions: The present analysis provides a basis for modeling homologous family 8 cellulases. The architecture of the active-site cleft, presenting at least five glucosyl-binding subsites, explains why family 8 cellulases cleave cello-oligosaccharide polymers that are at least five D-glycosyl subunits long. Furthermore, the structure of CelA allows comparison with (alpha/alpha)6 barrel glycosidases that are not related in sequence, suggesting a possible, albeit distant, evolutionary relationship between different families of glycosyl hydrolases.

The structure of Clostridium thermocellum endoglucanase CelC, a member of the largest cellulase family (family A), has been determined at 2.15 A resolution. The protein folds into an (alpha/beta)8 barrel, with a deep active-site cleft generated by the insertion of a helical subdomain. The structure of the catalytic core of xylanase XynZ, which belongs to xylanase family F, has been determined at 1.4 A resolution. In spite of significant differences in substrate specificity and structure (including the absence of the helical subdomain), the general polypeptide folding pattern, architecture of the active site and catalytic mechanism of XynZ and CelC are similar, suggesting a common evolutionary origin.

The crystal structure of Clostridium thermocellum endoglucanase CelD revealed an extended NH2-terminal segment (involving residues from the putative leader peptide) sticking out from the enzyme core to interact with a symmetry related molecule through an intermolecular salt bridge (Lys38-Asp201). Enzymatic digestion of CelD with various proteases emphasized the flexibility of the NH2-segment in solution. Proteolytic removal of Lys38 or the substitution of bridge-forming residues by site-directed mutagenesis promoted crystal packing arrangements that differ from that of wild type CelD. Crystals of wild-type CelD (a = 99.3 A c = 191.8 A) are trigonal, space group P3(1)21, with one molecule in the asymmetric unit (form A), whereas crystals of papain-treated CelD (a = 100.4 A, c = 248.7 A), of CelDK38M (a = 100.1 A, c = 248.4 A) and of papain-treated CelDD201A (a = 99.9 A, c = 250.0 A) are trigonal, space group P3(1)21, with two crystallographically independent molecules (form B), and crystals of chymotrypsin-treated CelD (a = 100.0 A, c = 254.3 A) and of CelDD201A (a = 99.8 A, c = 254.7 A) are hexagonal, space group P6(1)22, with one molecule in the asymmetric unit (form C). Only chymotrypsin-treated CelD (which preserves both Lys38 and Asp201) can grow in crystal form A upon macroseeding, indicating that formation of the intermolecular salt bridge is critical for stability of this crystal form. Flexible NH2- and COOH-terminal peptide extensions were found to influence crystal nucleation, but not crystal growth. The crystal structures of papain-treated CelD and chymotrypsin-treated CelD, determined at 3.5 A resolution by molecular replacement techniques, demonstrate that a small change in molecular orientation promoted by Lys38 account for the differences between crystal forms B and C.

The three-dimensional structures of the free and antigen-complexed Fabs from the mouse monoclonal anti-hen egg white lysozyme antibody D44.1 have been solved and refined by X-ray crystallographic techniques. The crystals of the free and lysozyme-bound Fabs were grown under identical conditions and their X-ray diffraction data were collected to 2.1 and 2.5 A, respectively. Two molecules of the Fab-lysozyme complex in the asymmetric unit of the crystals show nearly identical conformations and thus confirm the essential structural features of the antigen-antibody interface. Three buried water molecules enhance the surface complementarity at the interface and provide hydrogen bonds to stabilize the complex. Two hydrophobic buried holes are present at the interface which, although large enough to accommodate solvent molecules, are void. The combining site residues of the complexed FabD44.1 exhibit reduced temperature factors compared with those of the free Fab. Furthermore, small perturbations in atomic positions and rearrangements of side-chains at the combining site, and a relative rearrangement of the variable domains of the light (VL) and the heavy (VH) chains, detail a Fab accommodation of the bound lysozyme. The amino acid sequence of the VH domain, as well as the epitope of lysozyme recognized by D44.1 are very close to those previously reported for the monoclonal antibody HyHEL-5. A feature central to the FabD44.1 and FabHyHEL-5 complexes with lysozyme are three salt bridges between VH glutamate residues 35 and 50 and lysozyme arginine residues 45 and 68. The presence of the three salt bridges in the D44.1-lysozyme interface indicates that these bonds are not responsible for the 1000-fold increase in affinity for lysozyme that HyHEL-5 exhibits relative to D44.1.

Endoglucanase CelC from Clostridium thermocellum expressed in Escherichia coli has been crystallized in two different crystal forms by the hanging drop method. Crystals of form I were grown with polyethylene glycol as a precipitant. They are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 51.4 A, b = 84.3 A, and c = 87.5 A. Crystals of form II, obtained in ammonium sulfate solutions, belong to the tetragonal space group P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions of a = b = 130.7 A and c = 69.6 A. Diffraction data to 2.8 A resolution were observed for both crystal forms with a rotating anode generator. Preliminary oscillation images of the orthorhombic form I crystals using a synchrotron radiation source show diffraction to 2.2 A resolution, indicating that these crystals are suitable for high resolution crystallographic analysis.

The crystal structures of pheasant and guinea fowl lysozymes have been determined by X-ray diffraction methods. Guinea fowl lysozyme crystallizes in space group P6(1)22 with cell dimensions a = 89.2 A and c = 61.7 A. The structure was refined to a final crystallographic R-factor of 17.0% for 8,854 observed reflections in the resolution range 6-1.9 A. Crystals of pheasant lysozyme are tetragonal, space group P4(3)2(1)2, with a = 98.9 A, c = 69.3 A and 2 molecules in the asymmetric unit. The final R-factor is 17.8% to 2.1 A resolution. The RMS deviation from ideality is 0.010 A for bond lengths and 2.5 degrees for bond angles in both models. Three amino acid positions beneath the active site are occupied by Thr 40, Ile 55, and Ser 91 in hen, pheasant, and other avian lysozymes, and by Ser 40, Val 55, and Thr 91 in guinea fowl and American quail lysozymes. In spite of their internal location, the structural changes associated with these substitutions are small. The pheasant enzyme has an additional N-terminal glycine residue, probably resulting from an evolutionary shift in the site of cleavage of prelysozyme. In the 3-dimensional structure, this amino acid partially fills a cleft on the surface of the molecule, close to the C alpha atom of Gly 41 and absent in lysozymes from other species (which have a large side-chain residue at position 41: Gln, His, Arg, or Lys). The overall structures are similar to those of other c-type lysozymes, with the largest deviations occurring in surface loops. Comparison of the unliganded and antibody-bound models of pheasant lysozyme suggests that surface complementarity of contacting surfaces in the antigen-antibody complex is the result of local, small rearrangements in the epitope. Structural evidence based upon this and other complexes supports the notion that antigenic variation in c-type lysozymes is primarily the result of amino acid substitutions, not of gross structural changes.

The catalytic domain of a thermostable xylanase from Clostridium thermocellum has been expressed in Escherichia coli and crystallized from a polyethylene glycol 2000 solution by the hanging drop method. Crystals belong to the triclinic space group P1 with cell dimensions a = 46.8 A, b = 50.8 A, c = 70.3 A, alpha = 100.7 degrees, beta = 83.8 degrees, gamma = 101.6 degrees, and two molecules in the unit cell. These crystals diffract X-rays to at least 1.8 A resolution and are suitable for high-resolution X-ray analysis.

The complex formed between the Fab fragment of a murine monoclonal antihen egg lysozyme antibody F9.13.7 and the heterologous antigen Guinea-fowl egg lysozyme has been crystallized by the hanging drop technique. The crystals, which diffract X-rays to 3 A resolution, belong to the monoclinic space group P2(1), with a = 83.7 A, b = 195.5 A, c = 50.2 A, beta = 108.5 degrees and have two molecules of the complex in the asymmetric unit. The three-dimensional structure has been determined from a preliminary data set to 4 A using molecular replacement techniques. The lysozyme-Fab complexes are arranged with their long molecular axes approximately parallel to the crystallographic unique axis. Fab F9.13.7 binds an antigenic determinant that partially overlaps the epitope recognized by antilysozyme antibody HyHEL10.

A number of specific Fab and Fv fragments and their complexes with antigens (avian lysozymes), haptens, and anti-idiotopic Fabs have been studied by immunochemical and crystallographic techniques. Antigen and antibody interact through closely complementary contacting surfaces, without major conformational changes. An idiotopic determinant of a monoclonal antibody is shown to include parts of most of its complementarity determining regions. The specificity of antigen recognition resides in the close complementarity of the antigenic determinant with the antibody combining site.

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.

The cationic protein isolated from the granules of neutrophilic polymorphonuclear leukocytes inhibited the aggregation of human platelets as induced by collagen, thrombin, ristocetin, aggregated IgG by approximately 50% and anti-C1q by 75% but had no effect on aggregation-induced by ADP.