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Individuals with rare kidney diseases account for 5–10% of people with chronic kidney disease, but constitute more than 25% of patients receiving kidney replacement therapy. The National Registry of Rare Kidney Diseases (RaDaR) gathers longitudinal data from patients with these conditions, which we used to study disease progression and outcomes of death and kidney failure.

Genetic variants in coagulation factor IX (FIX) are associated with hemophilia B, a rare bleeding disease. F9variants are widespread across the gene and were summarized in our FIX variant database introduced in 2013.

Purpose: Drawing on institutional theory, this study aims to analyse the regulation of executive remuneration as espoused in the United Kingdom (UK) codified corporate governance principles, focussing on sources of advice to decision-makers, the nature of the advice sought and given, and interaction of those involved in the process. Design/methodology/approach: A qualitative research design was used. Data were assembled from interviewing non-executive board/remuneration committee members; institutional investors; external remuneration consultants and internal human resources (HR)/reward specialists. Results were analysed in accordance with the Gioia technique. Findings: Tensions inherent in the interpretation of corporate governance codes are illustrated. Emphasis on independent advice combined with constraints on decision-makers' capacity to navigate the nuances of a complex field and reputational concerns risks standardised instead of bespoke remuneration approaches aligned with corporate contexts. Practical implications: There is a role for internal HR advisors to add value through their potential to reduce the gap within remuneration committees between institutional contexts and independent decision-making, facilitating more strategic human resource management inspired executive remuneration. Originality/value: Application of institutional theory indicates the relevance of balancing external with internal sources to secure advice that is horizontally and vertically aligned within an organisation to meet the letter and spirit of corporate governance norms. Extending the explanatory power of institutional theory, care is needed though not to overlook the normative underpinnings of professional advisors' own value sets.

Purpose: The purpose of this paper is to explore the social construction of executive pay in the UK via an examination of narratives drawn from the social actors on the front-line of Key Management Personnel (KMP) pay determination. Design/methodology/approach: The authors' qualitative research draws upon in-depth interviews with non-executive directors (NEDs) serving on remuneration committees, institutional investors, and independent pay consultants. Findings: Regulation, market pricing and risk mitigation together with the social processes inherent within discharging corporate governance responsibilities create a status-quo-preserving isomorphic effect, restricting context-sensitive approaches to KMP pay determination. Practical implications: The paper informs action by company directors, investors and policy makers to address KMP pay controversies, building shared accountability amongst decision-makers focussed on more strategic context-aligned processes and outcomes. Originality/value: The authors' analysis illustrates how institutional isomorphism can be applied to analyse social actors' interpretations within KMP pay decision-making. The authors show that normative, coercive and mimetic isomorphic forces must be applied in dynamic interaction to extend the explanatory power of institutional isomorphism through the creation of a “No-Come-In” effect in respect of contemporary KMP pay determination settings.

Purpose: The purpose of this paper is to examine how female expatriates interpret the effectiveness of practical implementation of equality/diversity policies, trusting this to support their expatriate careers. Design/methodology/approach: A cross-sectional, qualitative research approach draws upon in-depth semi-structured interviews with 14 human resources equality/diversity policy implementers and 26 current female expatriates in two oil and gas firms. Findings: Early-career stage female expatriates believe that equality/diversity policy implementation will support their international careers. At the most senior levels, women expatriates highlight unequal treatment breaching their trust in delivery of equality/diversity principles to support their expatriate career progression. Research limitations/implications: Longitudinal research is needed to assess how early-career women expatriates' willingness to trust in organisational equality/diversity principles alters as their careers progress, and the effects of any changing trust relations on their contributions to organisational strategic objectives. Larger senior female expatriate samples are needed to research links between trust relations and turnover. Practical implications: Organisations must weigh up benefits from using transparent expatriate selection processes versus less formal mechanisms, if informal processes are not to undermine espoused equality interventions. Unconscious bias training should form part of wide-ranging programmes to tackle discrimination. Senior managerial action with embedded accountability is needed. Originality/value: Exploring the rhetoric and reality of equality/diversity policy implementation on women comprising a minority expatriate group, this research demonstrates women expatriates' early-career trust in gender equality falls away as they first recognise and then accept diminishing female expatriate senior grade representation and the implications for their expatriate careers. Should turnover result, this could detrimentally affect organisational expatriate gender diversity objectives.

Small angle x-ray and neutron scattering are techniques that give solution structures for large macromolecules. The creation of physically realistic atomistic models from known high-resolution structures to determine joint x-ray and neutron scattering best-fit structures offers a, to our knowledge, new method that significantly enhances the utility of scattering. To validate this approach, we determined scattering curves for two human antibody subclasses, immunoglobulin G (IgG) 1 and IgG4, on five different x-ray and neutron instruments to show that these were reproducible, then we modeled these by Monte Carlo simulations. The two antibodies have different hinge lengths that connect their antigen-binding Fab and effector-binding Fc regions. Starting from 231,492 and 190,437 acceptable conformations for IgG1 and IgG4, respectively, joint x-ray and neutron scattering curve fits gave low goodness-of-fit Rfactors for 28 IgG1 and 2748 IgG4 structures that satisfied the disulphide connectivity in their hinges. These joint best-fit structures showed that the best-fit IgG1 models had a greater separation between the centers of their Fab regions than those for IgG4, in agreement with their hinge lengths of 15 and 12 residues, respectively. The resulting asymmetric IgG1 solution structures resembled its crystal structure. Both symmetric and asymmetric solution structures were determined for IgG4. Docking simulations with our best-fit IgG4 structures showed greater steric clashes with its receptor to explain its weaker FcγRI receptor binding compared to our best-fit IgG1 structures with fewer clashes and stronger receptor binding. Compared to earlier approaches for fitting molecular antibody structures by solution scattering, we conclude that this joint fit approach based on x-ray and neutron scattering data, combined with Monte Carlo simulations, significantly improved our understanding of antibody solution structures. The atomistic nature of the output extended our understanding of known functional differences in Fc receptor binding between IgG1 and IgG4.

Purpose: The purpose of this paper is to surface and discuss issues associated with employee performance appraisal as a multi-staged social interaction reportedly the butt of managerial dissatisfaction, especially when used to inform decisions around pay and other rewards. Design/methodology/approach: To substantiate the territory, existing management-based evidence from the published literature is curated and discussed to frame issues for investigation under the rubric of performance appraisal as an activity that may be understood as combining interaction between forms of administrative, social and psychologically oriented control. Primary evidence, drawn from recent research sponsored by the Chartered Institute of Personnel and Development, which combines data sets informed by a survey of HR specialists and a follow-on focus group, is then used to illustrate views on relevant themes across a sample of UK-based private, public and third sector organizations (n=715). Findings: A significant number of organizations apply performance appraisal approaches, somewhat mediated by sector and size, and in turn use the results to inform various forms of HRM decision making – in particular reward management. While claims have been circulating in popular media suggesting the widespread abandonment of traditional performance appraisal, and while the study finds dissatisfaction regarding the utility of existing bureaucratic elements of appraisal mechanisms, the position is more nuanced. Practical implications: Corporate management attention is drawn to choices of the extent to which they are investing in building line management capabilities to address the consequences of policy decisions to amplify the importance of informal alongside formal performance management processes, and potential reward decision making, mindful of the indeterminate character of the employment relationship and its dynamic, socially constructed character. Social implications: Performance appraisal may benefit from re-interpreting the balance between emphasis on administrative, social and self-control, given changing expectations among workforce members and those who evaluate organizational effectiveness in contemporary society, and the ongoing contested nature of organizational control. Originality/value: Employee performance appraisal as an institutional process central to organizational control systems is a topic of interest to both organizational effectiveness academics and the managerial practitioners they study. Using data that broadly represent recent developments in managerial practice across “UK plc”, the paper informs reflection on theory and practice.

Purpose: The purpose of this paper is to reflect theoretically on a quarter-century of attempts to codify “best practice” standards related to oversight of and reporting on executive remuneration. Issues around the regulation of UK executive remuneration are analysed focussing on decision making by elite actors, informed by corporate governance codification artefacts and theoretical considerations inspired by notions of the social construction of reality. Design/methodology/approach: Using documentary materials to trace evolution of executive remuneration regulation in the UK, consideration is given to the social antecedents of processes governing corporate board remuneration committee practices. The paper reconstructs the social construction of the UK Corporate Governance Code and draws on relevant theoretically inclined literature to help make sense of processes involved. Findings: Shaping the problems, to be addressed as “legitimate problems”, is core to efforts intended to create “persuasive narratives” around how UK executive remuneration should be regulated. Research limitations/implications: The paper sketches an agenda for subsequent empirical “field” investigation to assess the social antecedents of UK executive remuneration outcomes. Practical implications: Offering an alternative way of thinking about executive reward and on-going controversy as to how it may be legitimately regulated, informed by contextual considerations. Originality/value: A novel look at executive remuneration from a social construction of reality perspective. Adding value to public debate on organisational effectiveness at a time of warnings from luminaries such as the Bank of England governor about the adverse social impact of “stateless companies” and calls for action against unfairness in income distribution.

X-ray and neutron-scattering techniques characterize proteins in solution and complement high-resolution structural studies. They are useful when either a large protein cannot be crystallized, in which case scattering yields a solution structure, or a crystal structure has been determined and requires validation in solution. These solution structures are determined by the application of constrained modelling methods based on known subunit structures. First, an appropriate starting model is generated. Next, its conformation is randomized to generate thousands of models for trial-and-error fits. Comparison with the experimental data identifies a small family of best-fit models. Finally, their significance for biological function is assessed. We illustrate this in application to structure determinations for secretory immunoglobulin A, the most prevalent antibody in the human body and a first line of defence in mucosal immunity. We also discuss the applications to the large multi-domain proteins of the complement system, most notably its major regulator factor H, which is important in age-related macular degeneration and renal diseases. We discuss the importance of complementary data from analytical ultracentrifugation, and structural studies of protein–protein complexes. We conclude that constrained scattering modelling makes useful contributions to our understanding of antibody and complement structure and function.

Antibodies and the complement proteins of our immune system are large highly glycosylated and flexible multidomain proteins whose solution structures and protein–protein interactions are intricate to unravel. Controversies and speculations abound in this field. The intact proteins are hard to crystallize, and far too big for NMR structural determinations. Thus alternative structural approaches based on neutron scattering and others such as X-ray scattering, constrained modelling and analytical ultracentrifugation become vital to tackle these proteins. The commissioning of the £145 million second target station (TS-2) project at ISIS represents a major advance that will offer much higher neutron beam fluxes and permit more dilute proteins to be studied and more ambitious experiments to be planned. Recently completed antibody projects at ISIS illustrate our present capabilities and provide tantalizing views of what will be achievable. ISIS has already made invaluable contributions to biochemistry, and will continue to do so with TS-2.

The structural properties of the linker peptide connecting the cellulose-binding module to the catalytic module in bimodular cellulases have been investigated by small-angle x-ray scattering. Since the linker and the cellulose-binding module are relatively small and cannot be readily detected separately, the conformation of the linker was studied by means of an artificial fusion protein, Cel6BA, in which an 88-residue linker connects the large catalytic modules of the cellulases Cel6A and Cel6B from Humicola insolens. Our data showed that Cel6BA is very elongated with a maximum dimension of 178Å, but could not be described by a single conformation. Modeling of a series of Cel6BA conformers with interdomain separations ranging between 10Å and 130Å showed that good Guinier and P(r) profile fits were obtained by a weighted average of the scattering curves of all the models where the linker follows a nonrandom distribution, with a preference for the more compact conformers. These structural properties are likely to be essential for the function of the linker as a molecular spring between the two functional modules. Small-angle x-ray scattering therefore provides a unique tool to quantitatively analyze the conformational disorder typical of proteins described as natively unfolded.

Anosmin-1, the gene product of the KAL gene, is implicated in the pathogenesis of X-linked Kallmann's syndrome. Anosmin-1 protein expression is restricted to the basement membrane and interstitial matrix of tissues affected in this syndrome during development. The anosmin-1 sequence indicates an N-terminal cysteine-rich domain, a whey acidic protein (WAP) domain, four fibronectin type III (FnIII) domains and a C-terminal histidine-rich region, and shows similarity with cell-adhesion molecules, such as neural cell-adhesion molecule, TAG-1 and L1. We investigated the structural and functional significance of three loss-of-function missense mutations of anosmin-1 using comparative modelling of the four FnIII and the WAP domains based on known NMR and crystal structures. Three missense mutation-encoded amino acid substitutions, N267K, E514K and F517L, were mapped to structurally defined positions on the GFCC′ β-sheet face of the first and third FnIII domains. Electrostatic maps demonstrated large basic surfaces containing clusters of conserved predicted heparan sulphate-binding residues adjacent to these mutation sites. To examine these modelling results anosmin-1 was expressed in insect cells. The incorporation of the three mutations into recombinant anosmin-1 had no effect on its secretion. The removal of two dibasic motifs that may constitute potential physiological cleavage sites for anosmin-1 had no effect on cleavage. Peptides based on the anosmin-1 sequences R254–K285 and P504–K527 were then synthesized in order to assess the effect of the three mutations on cellular adhesion, using cell lines that represented potential functional targets of anosmin-1. Peptides (10μg/ml) incorporating the N267K and E514K substitutions promoted enhanced adhesion to 13.S.1.24 rat olfactory epithelial cells and canine MDCK1 kidney epithelial cells (P<0.01) compared with the wild-type peptides. This result was attributed to the introduction of a lysine residue adjacent to the large basic surfaces. We predict that two of the three missense mutants increase the binding of anosmin-1 to an extracellular target, possibly by enhancing heparan sulphate binding, and that this critically affects the function of anosmin-1.

Factor H (FH) is a regulatory cofactor for the protease factor I in the breakdown of C3b in the complement system of immune defence, and binds to heparin and other polyanionic substrates. FH is composed of 20 short consensus/complement repeat (SCR) domains, for which the overall arrangement in solution is unknown. As previous studies had shown that FH can form monomeric or dimeric structures, X-ray and neutron scattering was accordingly performed with FH in the concentration range between 0.7 and 14 mg ml⁻¹. The radius of gyration of FH was determined to be 11.1-11.3 nm by both methods, and the radii of gyration of the cross-section were 4.4 nm and 1.7 nm. The distance distribution function P(r) showed that the overall length of FH was 38 nm. The neutron data showed that FH was monomeric with a molecular mass of 165,000(±17,000) Da. Analytical ultracentrifugation data confirmed this, where sedimentation equilibrium curve fits gave a mean molecular mass of 155,000(±3,000) Da. Sedimentation velocity experiments using the g*(s) derivative method showed that FH was monodisperse and had a sedimentation coefficient of 5.3(±0.1) S. In order to construct a full model of FH for scattering curve and sedimentation coefficient fits, homology models were constructed for 17 of the 20 SCR domains using knowledge of the NMR structures for FH SCR-5, SCR-15 and SCR-16, and vaccinia coat protein SCR-3 and SCR-4. Molecular dynamics simulations were used to generate a large conformational library for each of the 19 SCR-SCR linker peptides. Peptides from these libraries were combined with the 20 SCR structures in order to generate stereochemically complete models for the FH structure. Using an automated constrained fit procedure, the analysis of 16,752 possible FH models showed that only those models in which the 20 SCR domains were bent back upon themselves were able to account for the scattering and sedimentation data. The best-fit models showed that FH had an overall length of 38 nm and is flexible. This length is significantly less than a predicted length of 73 nm if the 20 SCR structures had been arranged in an extended arrangement. This outcome is attributed to several long linker sequences. These bent-back domain structures may correspond to conformational flexibility in FH and enable the multiple FH binding sites for C3 and heparin to come into close proximity. Copyright 2001 Academic Press

A case of a novel mutation in the F7gene that results in factor VII coagulant activity (VII:c) of less than 1% and VII antigen (VII:Ag) levels of 10% is presented. DNA analysis revealed a homozygous 15–base pair (bp) in-frame insertion-type mutation at nucleotide 10554. This insertion consisted of a duplication of residues leucine (L)213 to aspartic acid (D)217 (leucine, serine, glutamic acid, histidine, and aspartic acid), probably arising by slipped mispairing between 2 copies of a direct repeat (GCGAGCACGAC) separated by 4 bp. Molecular graphic analyses showed that the insertion is located at the surface of the catalytic domain in an exposed loop stabilized by extensive salt-bridge and hydrogen bond formation at which the calcium binding site is located. The mutation probably interferes with protein folding during VII biosynthesis and/or diminishes functional activity through the loss of calcium binding. In vitro expression studies demonstrated that the levels of VII:Ag in lysates of cells transfected with wild type VII (VIIWT) were equivalent to those with mutant type VII (VIIMT), but the level of secreted VIIMT was 5% to 10% that of VIIWT. Pulse chase studies demonstrated that VIIMT did not accumulate intracellularly, and studies with inhibitors of protein degradation showed that recombinant VIIMT was partially degraded in the pre-Golgi compartment. Accordingly, only small amounts of VIIMT with undetectable procoagulant activity were secreted into conditioned media. These results demonstrate that a combination of secretion and functional defects is the mechanism whereby this insertion causes VII deficiency.

Factor B is a key component of the alternative pathway of complement and is cleaved by factor D into the Ba and Bb fragments in the presence of activated C3 (C3b or C3(H2O)). The Ba fragment contains three short consensus/complement repeat domains, while the Bb fragment contains a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain, all three of which are implicated in multisite contacts with C3. The upfield-shifted signals in the 1H NMR spectra of factor B, the Ba and Bb fragments, and the vWF-A and SP domains were used as sensitive conformational probes of their structures. Temperature studies and pH titrations showed that the Ba fragment and the vWF-A and SP domains had conformationally mobile structures. The comparison of the NMR spectra of the SP domains of both factor B and factor D showed that the factor D linewidths were broader than those for factor B, which may result from a range of proteolytically inactive conformations of factor D in the absence of substrate. The NMR spectra from the separate vWF-A and SP domains in combination with that of the Ba fragment generally accounted for that of intact factor B, apart from the perturbation of an upfield-shifted signal from the Ba fragment. A new upfield-shifted signal was observed in the Bb fragment that was not detected in the spectra for the vWF-A or SP domains or intact factor B. Ring current calculations based on homology models or crystal structures predicted that buried hydrophobic methyl-aromatic interactions probably accounted for the upfield-shifted signals, with many arising from the N-terminal subdomain of the SP domain to which the C terminus of the vWF-A domain is directly linked. It was concluded that: (1) the conformation of the free SP domain is better ordered in solution than that of factor D; (2) the conformation of the Ba fragment is affected by its incorporation into factor B; and (3) the proximity of the vWF-A and SP domains within the Bb fragment leads to a conformational change in which conserved charged residues may be important. Allosteric structural rearrangements in the SP domain as the result of its interactions with the vWF-A domain or the Ba fragment provide an explanation of the regulation of the catalytic activity of factor B.

MFE23 is a single chain Fv antibody that has a high affinity for carcinoembryonic antigen (CEA). A full homology model for CEA based on V‐type, I‐type and C2‐type immunoglobulin folds, 28 oligosaccharides and the interdomain angle of CD2 was validated using solution scattering data. The superimposition of the intermolecular contacts observed in our recent crystal structure of MFE23 with the N‐terminal domain of CEA permitted the MFE23–CEA complex to be modelled. Good surface and electrostatic complementarity and carbohydrate‐unhindered access of MFE23 with the indentation between the first two CEA domains was observed. The model is supported by biochemical data and provides insight on the high affinity of MFE23 for CEA.

Factor B is a key component of the alternative pathway of complement and is cleaved by factor D into the Ba and Bb fragments when complexed with the activated form of C3, namely C3b. The Bb fragment contains a von Willebrand factor type A (vWF-A) domain, which is composed of an open twisted almost-parallel β-sheet flanked on both sides by seven α-helices A1 to A7, with a metal coordination site at its active-site cleft. Homology modelling of this vWF-A domain shows that the metal-binding site was present. Two recombinant vWF-A domains (Gly229-Ile444 and Gly229-Gln448) were examined by circular dichroism and Fourier transform infrared spectroscopy and indicated a significant conformational transition in the presence and absence of Mg2+. Two upfield-shifted signals in the 1H NMR spectrum were used as sensitive probes of the vWF-A protein structure, one of which was assigned to a methyl group and demonstrated metal- and pH-dependent properties between two distinct conformations. Temperature denaturation studies followed by spectroscopy showed that metal-binding caused the vWF-A structure to become significantly more stable. Ring current calculations based on a homology model for the vWF-A structure correlated one upfield-shifted signal with a methyl group on the α-helices in the vWF-A structure and the other one with individual single protons. An allosteric property of the vWF-A domain has thus been identified, and its implications for factor B activation were examined. Since the vWF-A domain after α-helix A7 is connected by a short link to the catalytic serine protease domain in the Bb fragment, the identification of a metal-free and a more stable metal-bound conformation for the vWF-A domain implies that the vWF-A interaction with C3b may alter its Mg2+-bound coordination in such a way as to induce conformational changes that may regulate the proteolytic activity of factor B.

MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly4Ser)3 linker to a variable light-chain (VL) domain (ĸ chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 Å resolution by molecular replacement with an Rcryst of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a β-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the β-sheet of the VL domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

Factor B is a five-domain 90 kDa serine protease proenzyme which is part of the human serum complement system. It binds to other complement proteins C3b and properdin, and is activated by the protease factor D. The fourth domain of factor B is homologous to the type A domain of von Willebrand Factor (vWF-A). A full-length human factor B cDNA clone was used to amplify the region encoding the vWF-A domain (amino acids 229-444 of factor B). A fusion protein expression system was then used to generate it in high yield in Escherichia coli, where thrombin cleavage was used to separate the vWF-A domain from its fusion protein partner. A second vWF-A domain with improved stability and solubility was created using a Cys267 → Ser mutation and a four-residue C-terminal extension of the first vWF-A domain. The recombinant domains were investigated by analytical gel filtration, sucrose density centrifugation and analytical ultracentrifugation, in order to show that both domains were monomeric and possessed compact structures that were consistent with known vWF-A crystal structures. This expression system and its characterization permitted the first investigation of the function of the isolated vWF-A domain. It was able to inhibit substantially the binding of 125I-labelled factor B to immobilized C3b. This demonstrated both the presence of a C3b binding site in this portion of factor B and a ligand-binding property of the vWF-A domain. The site at which factor D cleaves factor B is close to the N-terminus of both recombinant vWF-A domains. Factor D was shown to cleave the vWF-A domain in the presence or absence of C3b, whereas the cleavage of intact factor B under the same conditions occurs only in the presence of C3b.

The interaction between homologous DNA molecules in recombination and DNA repair leads to the formation of crossover intermediates known as Holliday junctions. Their enzymatic processing by the RuvABC system in bacteria involves the formation of a complex between RuvA and the Holliday junction. To study the solution structure of this complex, contrast variation by neutron scattering was applied to Mycobacterium lepraeRuvA (MleRuvA), a synthetic analogue of a Holliday junction with 16 base-pairs in each arm, and their stable complex. Unbound MleRuvA was octameric in solution, and formed an octameric complex with the DNA junction. The radii of gyration at infinite contrast were determined to be 3.65 nm, 2.74 nm and 4.15 nm for MleRuvA, DNA junction and their complex, respectively, showing that the complex was structurally more extended than MleRuvA. No difference was observed in the presence or absence of Mg2+. The large difference in RGvalues for the free and complexed protein in 65% 2H2O, where the DNA component is “invisible”, showed that a substantial structural change had occurred in complexed MleRuvA. The slopes of the Stuhrmann plots for MleRuvA and the complex were 19 and 15 or less (× 10−5), respectively, indicating that DNA passed through the centre of the complex. Automated constrained molecular modelling based on the Escherichia coliRuvA crystal structure demonstrated that the scattering curve of octameric MleRuvA in 65% and 100% 2H2O is explained by a face-to-face association of two MleRuvA tetramers stabilised by salt-bridges. The corresponding modelling of the complex in 65% 2H2O showed that the two tetramers are separated by a void space of about 1–2 nm, which can accommodate the width of B-form DNA. Minor conformational changes between unbound and complexed MleRuvA may occur. These observations show that RuvA plays a more complex role in homologous recombination than previously thought.

Human immunoglobulin A (IgA) is an abundant antibody that mediates immune protection at mucosal surfaces as well as in plasma. The IgA1 isotype contains two four-domain Fab fragments and a four-domain Fc fragment analogous to that in immunoglobulin G (IgG), linked by a glycosylated hinge region made up of 23 amino acid residues from each of the heavy chains. IgA1 also has two 18 residue tailpieces at the C terminus of each heavy chain in the Fc fragment. X-ray scattering using H2O buffers and neutron scattering using 100 %2H2O buffers were performed on monomeric IgA1 and a recombinant IgA1 that lacks the tailpiece (PTerm455). The radii of gyration RGfrom Guinier analyses were similar at 6.11–6.20 nm for IgA1 and 5.84–6.16 nm for PTerm455, and their cross-sectional radii of gyration RXSwere also similar. The similarity of theRGand RXSvalues suggests that the tailpiece of IgA1 is not extended outwards in solution. The IgA1 RGvalues are higher than those for IgG, and the distance distribution function P(r) showed two distinct peaks, whereas a single peak was observed for IgG. Both results show that the hinge of IgA1 results in an extended Fab and Fc arrangement that is different from that in IgG. Automated curve-fit searches constrained by homology models for the Fab and Fc fragments were used to model the experimental IgA1 scattering curves. A translational search to optimise the relative arrangement of the Fab and Fc fragments held in a fixed orientation resembling that in IgG was not successful in fitting the scattering data. A new molecular dynamics curve-fit search method generated IgA1 hinge structures to which the Fab and Fc fragments could be connected in any orientation. A search based on these identified a limited family of IgA1 structures that gave good curve fits to the experimental data. These contained extended hinges of length about 7 nm that positioned the Fab-to-Fab centre-to-centre separation 17 nm apart while keeping the corresponding Fab-to-Fc separation at 9 nm. The resulting extended T-shaped IgA1 structures are distinct from IgG structures previously determined by scattering and crystallography which have Fab-to-Fab and Fab-to-Fc centre-to-centre separations of 7–9 nm and 6–8 nm, respectively. It was concluded that the IgA1 hinge is structurally distinct from that in IgG, and this results in a markedly different antibody structure that may account for a unique immune role of monomeric IgA1 in plasma and mucosa.

Factor B is a key component of the alternative pathway of the complement system. During complement activation, factor B complexed with activated C3 is cleaved into the Ba and Bb fragments by the protease factor D to form the C3 convertase from the complex between C3b and Bb. The Ba fragment contains three short consensus/complement repeat (SCR) domains, and the Bb fragment contains a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain. Surface-enhanced laser desorption-ionization affinity mass spectrometry (SELDIAMS) was used to investigate the reaction of factor B with immobilised activated C3(NH 3) in the presence of Mg2+. A recombinant vWF-A domain (residues G229-Q448), the native Ba and Bb fragments and native factor B all demonstrated specific interactions with C3(NH3), while no interactions were detected using bovine serum albumin as a control. A mass analysis of the proteolysis of the vWF-A domain when this was bound to immobilised C3(NH3) identified two peptides (residues G229-K265 and T355-R381) that were involved with vWF-A binding to C3(NH 3). A homology model for the vWF-A domain was constructed using the vWF-A crystal structure in complement receptor type 3. Comparisons with five different vWF-A crystal structures showed that large surface insertions were present close to the carboxyl and amino edges of the central β-sheet of the factor B vWF-A structure. The peptides G229-K265 and T355-R381 corresponded to the two sides of the active site cleft at the carboxyl edge of the vWF-A structure. The vWF-A connections with the SCR and SP domains were close to the amino edge of this vWF-A β-sheet, and shows that the vWF-A domain can be involved in both C3b binding and the regulation of factor B activity. These results show that (i) a major function of the vWF-A domain is to bind to activated C3 during the formation of the C3 convertase, which it does at its active site cleft; and that (ii) SELDIAMS provides an efficient means of identifying residues involved in protein-protein interactions.

von Willebrand factor (vWF) is a large multimeric, multidomain glycoprotein found in platelets, endothelial cells and plasma. The A1, A2, and A3 domains in vWF mediate binding to glycoprotein Ib, ristocetin, botrocetin, collagen, sulphatides, and heparin and provide a protease cleavage site. Mutations causing types 2B, 2M, and 2A von Willebrand's disease (vWD) are located in the A1 and A2 domains. Homology modeling was performed to provide a molecular interpretation of vWF function and mutation sites. This was based on our previous alignment of 75 vWF-A sequences, the doubly wound a/ß fold seen in recent vWF-A crystal structures from complement receptor type 3 and lymphocyte function-associated antigen-1, and our new alignment of 28 vWF A1 and A2 sequences from different species. The active site in doubly-wound a/ß folds forms a crevice that is located at the switch point between the two halves of the central ß-sheet, and usually contains two metal-binding Asp residues in the vWF-A superfamily. Although one of these Asp residues is absent from the A1, A2, and A3 domains, this crevice is shown to correspond to the ristocetin binding site in the A1 domain and the protease cleavage site in the A2 domain. The residues R571-K572-R578-R579-K585 are found to be conserved in 28 A1 sequences and are predicted to constitute the heparin binding site in the A1 domain. Inspection of the type 2M vWD mutation sites that are involved in downregulation of glycoprotein Ib (GpIb) binding to vWF shows that these are spatially clustered at the carboxyl-edge of the ß-sheet and above it in the A1 domain and may directly perturb GpIb binding. In contrast, the type 2B vWD mutation sites that are involved in upregulation of GpIb binding to vWF are spatially clustered at the amino edge of this ß-sheet and below it and are located on the opposite side of the A1 domain from the type 2M mutation sites. The type 2B mutations are located between the heparin and GpIb binding sites. Because heparin binding inhibits the interaction with GpIb, this provides an explanation of vWF upregulation. The type 2A vWD mutation sites in the A2 domain correspond to buried residues that are otherwise 100% conserved across all 28 species, and are likely to be important for the correct folding of the A2 domain and its physiologically important protease site.

Human serum amyloid P component (SAP) is a normal plasma glycoprotein and the precursor of amyloid P component which is a universal constituent of the abnormal tissue deposits in amyloidosis. X-ray and neutron scattering data showed that pentameric or decameric ring structures for SAP in solution are readily distinguished. Further neutron data collection showed that SAP pentamers were reproducibly obtained in the presence of Ca2+at pH 5.5 or in the presence of methyl 4,6-O-(1-carboxyethylidene)-β-d-galactopyranoside (MOβDG) and Ca2+at pH 6.0 to 8.0, while SAP decamers were obtained in the presence of EDTA between pH 5.5 and 8.0. SAP pentamers have a mean X-ray RGof 3.99(±0.11) nm and a mean neutron RGof 3.69(±0.12) nm in 100% 2H2O. SAP decamers have a mean X-ray RGof 4.23(±0.12) nm and a mean neutron RGof 4.09(±0.14) nm in 100% 2H2O. The absorption coefficients of SAP pentamers and decamers differ by 10%. If we infer that the two α-helical A-faces are in contact with each other in the SAP decamer, the lack of structural change of the decamer with pH may be explained by the absence of His residues from the A-face of the SAP pentamer, and the change in absorption coefficients is compatible with the presence of Trp residues at this A-face. The rigid ring structure of pentameric SAP provided a test of scattering curves calculated from crystal structures. The only structural unknown is the orientation of the five chemically homogeneous oligosaccharide chains relative to the protein, but extended oligosaccharide structures were found to account for its scattering curve. X-ray scattering curves were best calculated using a hydrated structure, while neutron scattering curves were best calculated using an unhydrated structure. The outcome of these analyses was used to model the structure of decameric SAP. The evaluation of 640 structures for two SAP pentamers brought face-to-face to form SAP decamers gave better curve fits for structures in which the two A-faces were in contact with each other, in which it is likely that the two pentamers were out of alignment by a rotation of 36° and the oligosaccharide chains were extended.

Link protein and aggrecan of the extracellular matrix each contain two proteoglycan tandem repeat (PTR) domains that interact with hyaluronate. Consensus secondary structure predictions for 59 PTR sequences and 129 C‐type lectin sequences give similar patterns of two a‐helices and up to seven β‐strands. Protein fold recognition analyses show that the 59 PTR sequences are highly compatible with the C‐type lectin crystal structure. The predicted fold consists of a conserved motif formed from an antiparallel β‐sheet flanked by two α‐helices, the motif being attached to two distinct types of sheet region in the two superfamilies. Arg9 or Lys11 on an exposed loop and up to three other Arg residues in the β‐sheet region are conserved and may form part of a hyaluronate binding site.

The fold of the von Willebrand Factor type A domain (vWF-A) was predicted to be similar to an α/β doubly wound fold in the GTP-binding domain of ras-p21, despite the lack of sequence or functional similarity. This was subsequently confirmed by the vWF-A crystal structure from complement receptor type 3. The prediction is now reviewed. The vWF-A secondary structure was predicted with 62 to 75% accuracy and 12 of the 13 secondary structure elements were identified correctly. Accessibility predictions were 69 to 71% accurate. The fold recognition analysis was confirmed, but was much improved by averaging the results from 70 complete vWF-A sequences. The related folds of ras-p21 and flavodoxin scored highly. In addition, both the mapping of the predicted vWF-A secondary structure elements with those in 12 known α/β folds and two Asp residues at the C-terminal ends of two adjacent β-strands matched well with ras-p21 and flavodoxin. The predicted Mg^{2 +}-binding site, two disulphide bridges and the secondary structure topology were largely accurate. The exception is the reversal of a β-hairpin at one end of the central β-sheet. We conclude that non-homologous folds with dissimilar functions can be predicted from sequence data with reasonable accuracy, and that the accuracy in this case was principally limited at the periphery of the fold.

Low density lipoprotein receptor domains (LDLrs) represent a large cell surface receptor superfamily of consensus length 39 residues. Alignment of 194 sequences indicated highly conserved Cys and Asp/Glu residues, and a consensus secondary structure with three β-strands was predicted. Sequence threading against known protein folds indicated consistency with small β-sheet proteins. Complement factor I contains two LDLrs, and the second of these was successfully expressed using a bacterial pGEX system. FT-IR spectroscopy on this indicated a small amount of β-sheet together with turns and loops. LDLr is proposed to have a β-sheet structure in which the five biologically important Asp/Glu residues are located on an exposed loop.

Carcinoembryonic antigen (CEA) is one of the most widely used cell-surface tumour markers for tumour monitoring and for targeting by antibodies. It is heavily glycosylated (50% carbohydrate) and a monomer is constructed from one V-type and six C2-type fold domains of the immunoglobulin superfamily. The solution arrangement at low resolution of the seven domains in CEA cleaved from its membrane anchor was determined by X-ray and neutron scattering. Guinier analyses showed that the X-ray radius of gyrationR_Gof CEA was 8.0 nm. The length of CEA was 27 to 33 nm, and is consistent with an extended arrangement of seven domains. The X-ray cross-sectional radius of gyrationR_XSwas 2.1 nm, and is consistent with extended carbohydrate structures in CEA. The neutron data gave CEA a relative molecular mass of 150,000, in agreement with a value of 152,500 from composition data, and validated the X-ray analyses. The CEA scattering curves were analysed using an automated computer modelling procedure based on the crystal structure of CD2. The V-type and C2-type domains in CD2 were separated, and the C2-type domain was duplicated five times to create a linear seven-domain starting model for CEA. A total of 28 complex-type oligosaccharide chains in extended conformations were added to this model. By fixing the six interdomain orientations to be the same, three-parameter searches of the rotational orientations between the seven domains gave 4056 possible CEA models. The best curve fits from these corresponded to a family of zig-zag models. The long axis of each domain was set at 160(±25)° relative to its neighbour, and the two perpendicular axes were orientated at 10(±30)° and −5(±35)°. Interestingly, the curve fit from this model is within error of that calculated from a CEA model generated directly from the CD2 crystal structure by the superposition of adjacent domains. Zig-zag models of this type imply that the protein face of the GFCC' β-sheet in neighbouring CEA domains lie on alternate sides of the CEA structure. Such a model has implications for the adhesion interactions between CEA molecules on adjacent cells or for the antibody targeting of CEA.

The E7 and E6 proteins are the main oncoproteins of human papillomavirus types 16 and 18 (HPV-16 and HPV-18), and possess unknown protein structures. E7 interacts with the cellular tumour-suppressor protein pRB and contains a zinc-binding site with two Cys-Xaa2-Cys motifs spaced 29 or 30 residues apart. E6 interacts with another cellular tumour-suppressor protein p53 and contains two zinc-binding sites, each with two Cys-Xaa2-Cys motifs at a similar spacing of 29 or 30 residues. By using the GOR I/III, Chou-Fasman, SAPIENS and PHD methods, the effectiveness of consensus secondary structure predictions on zinc-finger proteins was first tested with sequences for 160 transcription factors and 72 nuclear hormone receptors. These contain Cys2His2 and Cys2Cys2 zinc-binding regions respectively, and possess known atomic structures. Despite the zinc- and DNA-binding properties of these protein folds, the major α-helix structures in both zinc-binding regions were correctly identified. Thus validated, the use of these prediction methods with 47 E7 sequences indicated four well-defined α-helix (α) and β-sheet (β) secondary structure elements in the order ββαβ in the zinc-binding region of E7 at its C-terminus. The prediction was tested by Fourier transform infrared spectroscopy of recombinant HPV-16 E7 in H2O and 2H2O buffers. Quantitative integration showed that E7 contained similar amounts of α-helix and β-sheet structures, in good agreement with the averaged prediction of α-helix and β-sheet structures in E7 and also with previous circular dichroism studies. Protein fold recognition analyses predicted that the structure of the zinc-binding region in E7 was similar to a ββαβ motif found in the structure of Protein G. This is consistent with the E7 structure predictions, despite the low sequence similarities with E7. This predicted motif is able to position four Cys residues in proximity to a zinc atom. A model for the zinc-binding motif of E7 was constructed by combining the Protein G coordinates with those for the zinc-binding site in transcription factor TFIIS. Similar analyses for the two zinc-binding motifs in E6 showed that they have different α/β secondary structures from that in E7. When compared with 12 other zinc-binding proteins, these results show that E7 and E6 are predicted to possess novel types of zinc-binding structure.

Low-density lipoproteins (LDLs) in plasma are constructed from a single molecule of apolipoprotein B-100 (apoB) (Mr 512000) in association with lipid [approximate Mr (2–3)×106]. LDL oxidation is an important process in the development of atherosclerosis, and can be imitated by the addition of Cu2+ ions. Synchrotron X-ray scattering of LDL yields curves without radiation damage effects at concentrations close to physiological. The radius of gyration RG for preparations of LDL from different donors ranged between 12.1 and 16.0 nm, with a mean of 13.9 nm. At 4 °C, the distance distribution curve P(r) indicated a maximum dimension of 25–27 nm for LDL, a peak at 19.5 nm which corresponds to a surface shell of protein and phospholipid head groups in LDL, and submaxima between 1.7 and 13.5 nm, which correspond to an ordered lipid core in LDL. LDL from different donors exhibited distinct P(r) curves. For oxidation studies of LDL by X-rays, data are best obtained at 4 °C at a concentration of ⩾ 2 mg of LDL protein/ml together with controls based on non-oxidized LDL. LDL oxidation (2 mg of apoB/ml) was studied at 37 °C in the presence of 6.4, 25.6 and 51.2 µmol of Cu2+/g of apoB. Large changes in P(r) were reproducibly observed in the inter-particle distance range between 13 and 16 nm shortly after initiation of oxidation. This corresponds to the phospholipid hydrocarbon in LDL, which has either increased in electron density during oxidation or become increasingly disordered. After 25 h, the structural changes subsequently spread to regions of the P(r) curves assigned to surface apoB and the central core of cholesteryl esters and triacylglycerols. Lipid analyses were carried out under the same solution conditions. The α-tocopherol and β-carotene antioxidant contents of LDL were consumed within 1–2 h. Analyses of the formation of thiobarbituric acid-reactive substances and lipid hydroperoxides indicated that arachidonic acid was preferentially oxidized before the maximal formation of lipid hydroperoxides at 8–12 h after initiation of oxidation. High-performance TLC showed that phosphatidylcholine was continuously converted into lysophosphatidylcholine during oxidation, which is consistent with the early changes in the X-ray P(r) curves. The neutral core lipids became modified only after 12–15 h of oxidation. The combination of X-ray scattering structural analyses with biochemical analyses shows that the oxidation of LDL first affects the outer shell of surface phospholipid, then it spreads towards damage of apoB and the internal neutral lipid core of LDL.

The type A domain of the von Willebrand Factor is found also in the complement proteins factor B (FB), C2, CR3 and CR4, the integrins, collagen types VI, VII, XII and XIV, and other proteins. FB is a component of the alternative pathway of the complement system of immune defence, and is cleaved into the fragments Bb and Ba during complement activation. Bb contains a von Willebrand Factor type A (vWF) domain of unknown secondary structure and a serine proteinase (SP) domain, whereas Ba contains three short consensus repeat/complement control protein (SCR/CCP) domains. Fourier transform infrared (FT-IR) spectroscopy on a recombinant vWF domain and on FB and its Bb and Ba fragments shows a broad amide I band. In h₂O buffer, second derivative spectra of the amide I band show subcomponents at 1654 to 1657 cm^-1, which is typical of α-helix, and at 1676 to 1685 cm^-1 and 1636 to 1637 cm^-1, which are typical of β-strand. α-Helix was detected in the vWF domain, the Bb fragment and FB, and the proportion of α-helix present decreased in that order. This shows that the vWF domain contains appreciable amounts of α-helix, while the SP and SCR/CCP domains are almost entirely β-sheet in their secondary structures. Quantitative integration of the vWF FT-IR spectrum showed that this contained 31% α-helix and 36% β-sheet. In ²H₂O buffer, the α-helix content in the vWF domain is sensitive to the solvent, while the β-sheet content is less so. An alignment of 75 vWF type A sequences from 25 proteins was used for averaged secondary structure predictions of the total length of 206 residues by the Robson and Chou-Fasman methods. In support of the FT-IR analysis, a total of at least five well-predicted α-helices (35% of residues) and at least five well-predicted β-strands (21% of residues) were identified by both predictive methods, all of which were interspersed by regions of coil or turn conformations. Eight of the ten predicted α-helices and β-strands form an alternating arrangement with each other. Since the predicted α-helices are mostly amphipathic, and since the α-helix FT-IR band is sensitive to solvent, the α-helices are inferred to be on the protein surface. The predicted β-strands are hydrophobic and therefore inferred to be buried in the protein core, again in accordance with the FT-IR data. Putative glycosylation sites are found mainly in solvent-exposed positions in the predicted coil or turn regions (63%) and on α-helices (22%). Two Asp residues implicated in metal binding are located in two predicted hydrophilic loops just after the C-terminal end of two β-strands. Copyright 1994, 1999 Academic Press

The C1̄ inhibitor of the complement cascade forms stoichiometric complexes with C1̄r and C1̄s and controls the activation of first component C1 of complement. Literature sedimentation coefficients s° 20.wfor the complexes formed between C1̄ inhibitor, C1̄r and C1̄s were analysed using frictional ratios and the hydrodynamic sphere approach. A head-and-tail two-domain model for C1 inhibitor was combined with cylindrical hydrodynamic models for the six-domain structures of C1̄r and C1̄s. The hydrodynamic data show that the heavily glycosylated N-terminal domain of C1̄ inhibitor is positioned close to the two complement ‘short consensus repeat’ domains found in the centre of C1̄r and C1̄s.

The von Willebrand Factor type A domain is the prototype for a protein superfamily. It possesses no significant sequence similarity to any known protein structure. Secondary structure predictions indicate a largely alternating pattern of six α‐helices and six β‐strands. A protein fold for this domain is proposed to correspond to a doubly‐wound open twisted β‐sheet structure flanked by α‐helices. Close agreement was found with the GTP‐binding domain of human ras‐p21, provided that an extra α‐helix was inserted. The structure of the predicted fold showed high compatibility with the proximate location of two Mg2+‐binding Asp residues, two disulphide‐bridged Cys residues, and other known functional attributes of this domain.