Human Immune Protein C1q Selectively Disaggregates Carbon Nanotubes.

We atomistically compute the change in free energy upon binding of the globular domain of the complement protein C1q to carbon nanotubes (CNTs) and graphene in solution. Our modeling results imply that C1q is able to disaggregate and disperse bundles of large diameter multiwalled CNTs but not those of thin single-walled CNTs, and we validate this prediction with experimental observations. The results support the view of a strong binding with potential implications for the understanding of the immune response and biomedical applications of graphitic nanomaterials.

Ficolins: innate immune recognition proteins for danger sensing

Las ficolinas son proteínas de defensa que forman oligómerosa partir de tallos homólogos al colágeno y dominios semejantesa fibrinógeno. Son capaces de sentir señales de peligro talescomo patrones moleculares asociados patógenos o a células apoptóticas.En el hombre, las ficolinas L y H se han caracterizado enel suero, mientras que la ficolina M es secretada por células monocíticas.Al igual que la lectina de unión a manano (“mannan-bindinglectin”, MBL), pueden asociarse a las serina-proteasas asociadasa MBL e iniciar la vía de activación de complemento de laslectinas, un importante sistema efector de la inmunidad innatahumoral. También pueden actuar como opsoninas, incrementandola eliminación de sus dianas por fagocitosis. Estudios estructuralesrecientes muestran que la ficolina L es una proteína dereconocimiento versátil, capaz de unir moléculas acetiladas y carbohidratosneutros por medio de sitios de unión diferentes, mientrasque la ficolina H posee un único sitio de unión con una especificidadmás restringida hacia los carbohidratos neutros. Los estudiosfilogenéticos revelan que las ficolinas han sido conservadasen el proceso evolutivo, apoyando la hipótesis de que el sistemade complemento primitivo era un sistema de opsonización basadoen lectinas, y ponen de relieve la importancia de las proteínasde reconocimiento de carbohidratos en la inmunidad innata

Ficolins are oligomeric defence proteins assembled from collagen-like stalks and fibrinogen-like domains that are able to sensedanger signals such as pathogen- or apoptotic cell-associated molecularpatterns. In humans, L- and H-ficolins have been characterizedin serum whereas M-ficolin is secreted by monocytic cells.Like mannan-binding lectin (MBL), they are able to associate withMBL-associated serine proteases and to trigger activation of thelectin pathway of complement, a major effector system of humoralinnate immunity. They can also act as opsonins to enhance clearanceof their targets by phagocytosis. Recent structural studieshave shown that L-ficolin is a versatile recognition protein ableto bind acetylated molecules and neutral carbohydrates throughdifferent binding sites, whereas H-ficolin has a single binding sitewith a more restricted specificity for neutral carbohydrates. Phylogeneticstudies reveal that ficolins have been conserved throughevolution, supporting the hypothesis that the primitive complementsystem was a lectin-based opsonic system, and emphasizingthe essential role of carbohydrate recognition proteins in innateimmunity

Structural biology of C1.

The classical complement pathway is a major element of innate immunity against infection, and is also involved in immune tolerance, graft rejection and various pathologies. This pathway is triggered by C1, a multimolecular protease formed from the association of a recognition protein, C1q, and a catalytic subunit, the calcium-dependent tetramer C1s-C1r-C1r-C1s, which comprises two copies of each of the modular proteases C1r and C1s. All activators of the pathway are recognized by the C1q moiety of C1, a process that generates a conformational signal that triggers self-activation of C1r, which in turn activates C1s, the enzyme that mediates specific cleavage of C4 and C2, the C1 substrates. Early work based on biochemical and electron microscopy studies has allowed characterization of the domain structure of the C1 subcomponents and led to a low-resolution model of the complex in which the elongated C1s-C1r-C1r-C1s tetramer folds into a compact, figure-of-8-shaped conformation upon interaction with C1q. The strategy used over the past decade was based on a dissection of the C1 proteins into modular segments to characterize their function and solve their three-dimensional structure by X-ray crystallography or NMR spectroscopy. This approach allows deep insights into the structure-function relationships of C1, particularly with respect to the assembly of the C1 complex and the mechanisms underlying its activation and proteolytic activity.

Structural biology of C1: dissection of a complex molecular machinery.

The classical pathway of complement is initiated by the C1 complex, a multimolecular protease comprising a recognition subunit (C1q) and two modular serine proteases (C1r and C1s) associated as a Ca2+-dependent tetramer (C1s-C1r-C1r-C1s). Early studies have allowed identification of specialized functional domains in these proteins and have led to low-resolution models of the C1 complex. The objective of current studies is to gain deeper insights into the structure of C1, and the strategy used for this purpose mainly consists of dissecting the C1 components into modular fragments, in order to solve their three-dimensional structure and establish the structural correlates of their function. The aim of this article is to provide an overview of the structural and functional information generated by this approach, with particular emphasis on the domains involved in the assembly, the recognition function, and the highly specific proteolytic properties of C1.

Crystal structure of the catalytic domain of human complement c1s: a serine protease with a handle.

C1s is the highly specific modular serine protease that mediates the proteolytic activity of the C1 complex and thereby triggers activation of the complement cascade. The crystal structure of a catalytic fragment from human C1s comprising the second complement control protein (CCP2) module and the chymotrypsin-like serine protease (SP) domain has been determined and refined to 1.7 A resolution. In the areas surrounding the active site, the SP structure reveals a restricted access to subsidiary substrate binding sites that could be responsible for the narrow specificity of C1s. The ellipsoidal CCP2 module is oriented perpendicularly to the surface of the SP domain. This arrangement is maintained through a rigid module-domain interface involving intertwined proline- and tyrosine-rich polypeptide segments. The relative orientation of SP and CCP2 is consistent with the fact that the latter provides additional substrate recognition sites for the C4 substrate. This structure provides a first example of a CCP-SP assembly that is conserved in diverse extracellular proteins. Its implications in the activation mechanism of C1 are discussed.

Structural and functional studies on C1r and C1s: new insights into the mechanisms involved in C1 activity and assembly.

C1r and C1s, the enzymes responsible for the activation and proteolytic activity of the C1 complex of complement, are modular serine proteases featuring similar overall structural organizations, yet expressing very distinct functional properties within C1. This review will initially summarize available information on the structure and function of the protein modules and serine protease domains of C1r and C1s. It will then focus on the regions of both proteases involved in: (i) assembly of C1s-C1r-C1r-C1s, the Ca(2+)-dependent tetrameric catalytic subunit of C1; (ii) expression of C1 catalytic activities. Particular emphasis will be aid on recent structural and functional studies that provide new insights into the complex mechanisms involved in the assembly, activation, and proteolytic activity of C1.

Evolutionary conserved rigid module-domain interactions can be detected at the sequence level: the examples of complement and blood coagulation proteases.

Several extracellular modular proteins, including proteases of the complement and blood coagulation cascades, are shown here to exhibit conserved sequence patterns specific for a particular module-domain association. This was detected by comparative analysis of sequence variability in different multiple sequence alignments, which provides a new tool to investigate the evolution of modular proteins. A first example deals with the proteins featuring a common complement control protein (CCP) module-serine protease (SP) domain pattern at their C-terminal end, defined here as the CCP-SP sub-family. These proteins include the complement proteases C1r, C1s and MASPs, the Limulus clotting factor C, and the proteins of the haptoglobin family. A second example deals with blood coagulation factors VII, IX and X and protein C, all featuring a common epidermal growth factor (EGF)-SP C-terminal assembly. Highly specific motifs are found at the connection between the CCP or EGF module and the activation peptide of the SP domain: [P/A]-x-C-x-[P/A]-[I/V]-C-G-x-[P/S/K] in the case of the CCP-SP proteins, and C-x-[P/S]-x-x-x-[Y/F]-P-C-G in the case of the EGF-SP proteins. Each motif is strictly conserved in the whole sub-family and it is detected in no more than one other known protein sequence. Strikingly, most of the conserved residues specific to each sub-family appear to be clustered at the interface between the SP domain and the CCP or EGF module. We propose that a rigid module-domain interaction occurs in these proteins and has been conserved through evolution. The functional implications of these assemblies, underlined by such evolutionary constraints, are discussed.

NMR structures of the C-terminal end of human complement serine protease C1s.

Synthetic peptides derived from the C-terminal end of the human complement serine protease C1s were analysed by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. Circular dichroism indicates that peptides 656-673 and 653-673 are essentially unstructured in water and undergo a coil-to-helix transition in the presence of increasing concentrations of trifluoroethanol. Two-dimensional NMR analyses performed in water/trifluoroethanol solutions provide evidence for the occurrence of a regular alpha-helix extending from Trp659 to Ser668 (peptide 656-673), and from Tyr656 to Ser668 (peptide 653-673), the C-terminal segment of both peptides remaining unstructured under the conditions used. Based on these and other observations, we propose that the serine protease domain of C1s ends in a 13-residue alpha-helix (656Tyr-Ser668) followed by a five-residue C-terminal extension. The latter appears to be flexible and is probably locked within C1s through a salt bridge involving Glu672.

Structure and assembly of the catalytic region of human complement protease C1r: a three-dimensional model based on chemical cross-linking and homology modeling.

C1r is the modular serine protease responsible for autocatalytic activation of C1, the first component of the complement classical pathway. Its catalytic region is a noncovalent homodimer of two gamma-B monomers, each comprising two contiguous complement control protein (CCP) modules, IV and V [also known as short consensus repeats (SCRs)], a 15-residue intermediary segment, and the serine protease B domain. With a view to gain insight into domain-domain interactions within this region, fragment C1r (gamma-B)2, obtained by autolytic proteolysis of the active protease, was cross-linked with the water-soluble reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Cross-linked species gamma-B intra and gamma-B inter, containing intra- and intermonomer cross-links, respectively, were isolated and then fragmented by CNBr cleavage and trypsin digestion. N-Terminal sequence and mass spectrometry analyses of the resulting cross-linked peptides allowed us to identify one intramonomer cross-link between Lys426 of module V and the C-terminal Asp688 of the serine protease B domain and one intermonomer cross-link between the N-terminal Gly280 of fragment gamma and Glu493 of the B domain. Three-dimensional homology modeling of the CCP modules IV and V and of the B domain was also performed. The complementary information provided by chemical cross-linking and homology modeling studies was used to construct a three-dimensional model of the gamma-B monomer, in which module V interacts with the serine protease on the side opposite to both the active site and the Arg446-Ile447 activation site. Also, a tentative three-dimensional model of the (gamma-B)2 dimer was built, indicating a loose "head to tail" association of the monomers, with the active sites facing opposite directions toward the outside of the dimer. The latter model is compared with available low-resolution structural data, and its functional implications are discussed in terms of the conformational changes occurring during C1r activation.

Crystal structure of human trypsin 1: unexpected phosphorylation of Tyr151.

The X-ray structure of human trypsin 1 has been determined in the presence of diisopropyl-phosphofluoridate by the molecular replacement method and refined at a resolution of 2.2 A to an R-factor of 18%. Crystals belong to the space group P4, with two independent molecules in the asymmetric unit packing as crystallographic tetramers. This study was performed in order to seek possible structural peculiarities of human trypsin 1, suggested by some striking differences in its biochemical behavior as compared to other trypsins of mammalian species. Its fold is, in fact, very similar to those of the bovine, rat and porcine trypsins, with root-mean-square differences in the 0.4 to 0.6 A range for all 223 C alpha positions. The most unexpected feature of the human trypsin 1 structure is in the phosphorylated state of tyrosine residue 151 in the present X-ray study. This feature was confirmed by mass spectrometry on the same inhibited sample and also on the native enzyme. This phosphorylation strengthens the outstanding clustering of highly negative or highly positive electrostatic surface potentials. The peculiar inhibitory behaviour of pancreatic secretory trypsin inhibitors of the Kazal type on this enzyme is discussed as a possible consequence of these properties. A charged surface loop has also been interpreted as an epitope site recognised by a monoclonal antibody specific to human trypsin 1.

How to escape from model bias with a high-resolution native data set - structure determination of the PcpA-S6 subunit III.

The structure of procarboxypeptidase A-S6 subunit III, a truncated zymogen E, has been determined by molecular replacement using as search model porcine elastase 1 which, as revealed by crystallographic analysis, contained about 20% of the amino acids in a radically different orientation. Two monoclinic crystal forms were used: the first one diffracts to 2.3 A resolution and contains one molecule per asymmetric unit; the second diffracts to 1.7 A resolution and contains two molecules per asymmetric unit. Molecular replacement and conventional X-PLOR refinement led to a model for which 20% of the chain was ill defined in both crystal forms. To remove the bias introduced by the initial model, an automated refinement procedure [Lamzin & Wilson (1993). Acta Cryst. D49, 129-147] was applied successfully to the second crystal form, which diffracts to high resolution. The resulting dramatic improvement of the electron-density map led to extensive rebuilding of some surface loops. The reliability of the modified model was confirmed by refinement of the first crystal form. For the two forms, the final R factor is 18.8% for data between 8.0 and 2.0 A resolution, and 18.4% for data between 8.0 and 1.7 A, respectively.

Structure of the catalytic region of human complement protease C1s: study by chemical cross-linking and three-dimensional homology modeling.

C1s is a multidomain serine protease that is responsible for the enzymatic activity of C1, the first component of the classical pathway of complement. Its catalytic region (gamma-B) comprises two contiguous complement control protein (CCP) modules, IV and V (about 60 residues each), a 15-residue intermediary segment, and the B chain (251 residues), which is the serine protease domain. With a view to identify domain-domain interactions within this region, the gamma-B fragment of C1s, obtained by limited proteolysis with plasmin, was chemically cross-linked with the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; then cross-linked peptides were isolated after CNBr cleavage and thermolytic digestion. N-Terminal sequence and mass spectrometry analyses allowed us to identify two cross-links between Lys 405 of module V and Glu 672 of the B chain and between Glu 418 of the intermediary segment and Lys 608 of the B chain. Three-dimensional modeling of the CCP modules IV and V and of the catalytic B chain was also carried out on the basis of their respective homology with the 16th and 5th CCP modules of complement factor H and type I serine proteases. The information provided by both the chemical cross-linking studies and the homology modeling enabled us to construct a three-dimensional model for the assembly of the C-terminal part of the gamma-B region, comprising module V, the intermediary segment, and the B chain. This model shows that module V interacts with the serine protease B chain on the side opposite to both the activation site and the catalytic site. Functional implications of this interaction are discussed in terms of the possible role of module V in the specific recognition and positioning of C4, one of the two substrates of C1s.

Crystal structure of bovine procarboxypeptidase A-S6 subunit III, a highly structured truncated zymogen E.

Subunit III, a defective serine endopeptidase lacking the typical N-terminal hydrophobic dipeptide is secreted by the pancreas of ruminant species as part of the bovine ternary complex procarboxypeptidase A-S6. Two monoclinic crystal forms were obtained and subsequently used to solve its X-ray structure. The highest resolution model of subunit III was refined at 1.7 A resolution to a crystallographic R-factor of 18.4%, with r.m.s. bond deviations from ideality of 0.012 A. About 80% of the model presents the characteristic architecture of trypsin-like proteases. The remaining zones, however, have well-defined, unique conformations. The regions from residues 70 to 80 and from 140 to 155 present maximum distances of 16 and 18 A relative to serine proteases and zymogens. Comparisons with the structures of porcine elastase 1 and chymotrypsinogen A indicate that the specific binding pocket of subunit III adopts a zymogen-like conformation and thus provide a basis for its inactivity. In general, the structural analysis of subunit III strongly suggests that it corresponds to a truncated version of a new class of highly structured elastase-like zymogen molecules. Based on the structures of subunit III and elastase 1, it is concluded that large concerted movements are necessary for the activation of zymogen E.

MANSEK and SUNHCA. Two interactive programs for the hydrophobic cluster analysis of protein sequences.

Hydrophobic cluster analysis (HCA) is an efficient method for analysing and comparing the amino acid sequences of proteins. It relies on two-dimensional representations of the sequences presently generated by simple plot programs working on microcomputers. Two interactive programs, MANSEK and SUNHCA, are described here that operate from Vax and Sun workstations respectively. These programs allow the display of several protein sequences in the form of two-dimensional helical plots suitable for HCA. Several tedious, repetitive and time-consuming steps of HCA have been suppressed by implementing several features such as interactive on-screen manipulations (zoom, translations) of the plots and HCA score calculations on segments chosen by the user. Plots on paper can be obtained through hard copies or plotting subroutines.

Hydrophobic cluster analysis reveals duplication in the external structure of human alpha-interferon receptor and homology with gamma-interferon receptor external domain.

Evidence is presented, based on sequence comparison according to Hydrophobic Cluster Analysis, of a structural and evolutionary relationship between the human alpha/beta-interferon receptor and the human and mouse gamma-interferon receptor. These results predict that the human alpha/beta-interferon receptor extracellular part is organised in two homologous subdomains connected by a proline linker. They also predict that both subdomains present some homologies to the external domain of mouse and human gamma interferon receptor.

Hydrophobic cluster analysis: procedures to derive structural and functional information from 2-D-representation of protein sequences.

Hydrophobic cluster analysis (HCA) [15] is a very efficient method to analyse and compare protein sequences. Despite its effectiveness, this method is not widely used because it relies in part on the experience and training of the user. In this article, detailed guidelines as to the use of HCA are presented and include discussions on: the definition of the hydrophobic clusters and their relationships with secondary and tertiary structures; the length of the clusters; the amino acid classification used for HCA; the HCA plot programs; and the working strategies. Various procedures for the analysis of a single sequence are presented: structural segmentation, structural domains and secondary structure evaluation. Like most sequence analysis methods, HCA is more efficient when several homologous sequences are compared. Procedures for the detection and alignment of distantly related proteins by HCA are described through several published examples along with 2 previously unreported cases: the beta-glucosidase from Ruminococcus albus is clearly related to the beta-glucosidases from Clostridum thermocellum and Hansenula anomala although they display a reverse organization of their constitutive domains; the alignment of the sequence of human GTPase activating protein with that of the Crk oncogene is presented. Finally, the pertinence of HCA in the identification of important residues for structure/function as well as in the preparation of homology modelling is discussed.

Crystallization and preliminary X-ray study of porcine trypsin, free and complexed with Ecballium elaterium trypsin inhibitor, a member of the squash inhibitors family.

Porcine trypsin has been crystallized either free or complexed with synthetic Ecballium elaterium trypsin inhibitor II, a 28-residue peptide with three disulfide bridges. The crystals diffract beyond 2.0 A. Crystals are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 77.32 A, b = 53.81 A, c = 46.91 A, for the free trypsin, and a = 62.25 A, b = 62.27 A, c = 84.66 A for the complex with E. elaterium trypsin inhibitor II.

Hydrophobic cluster analysis (HCA) of the hormone-binding domain of receptor proteins.

A new technique of protein sequence analysis, namely, Hydrophobic Cluster Analysis (HCA), has been used to align and compare the sequences of proteins belonging to the receptor superfamily (steroid, thyroid hormone and retinoic acid receptors) and serpin superfamily (corticosteroid binding globulin (CBG) and alpha 1-antitrypsin (alpha 1-AT]. By matching up clusters of hydrophobic amino-acids that oftenmost correspond to identifiable secondary structures (alpha-helices, beta-strands etc.), it has been possible to deduce the following information on the secondary structures of these proteins: CBG is structurally related to alpha 1-AT (HCA score greater than 80%), the structures of the hormone-binding domains of the steroid receptors that bind 3-keto-delta 4-steroids are closely interrelated (greater than 80%) but less closely related to that of the estrogen receptor (ER) (approximately 75%), vitamin D, retinoic acid and thyroid hormone receptors are structurally closely related (greater than or equal to 80%). Their secondary structures are, however, also related to that of the steroid receptors (approximately 70%), and a high degree of analogy exists between the structures of serpins and of the hormone-binding domains of members of the steroid superfamily (60-70%). HCA has clearly shown that a previous local sequence alignment of the estrogen receptor with other steroid receptors and cytochromes P450 has to be reconsidered. The published consensus steroid binding sequence previously identified in cytochromes is in fact 80 amino-acids upstream from its previously defined position. Other regions of contiguous sequence identity have also been identified which may be involved in the hydrophobic core of the protein or in steroid binding. Their positions have been indicated using the crystal structure of alpha 1-AT as a model.

Use of restrained molecular dynamics in water to determine three-dimensional protein structure: prediction of the three-dimensional structure of Ecballium elaterium trypsin inhibitor II.

Refinement of distance geometry (DG) structures of EETI-II (Heitz et al.: Biochemistry 28:2392-2398, 1989), a member of the squash family trypsin inhibitor, have been carried out by restrained molecular dynamics (RMD) in water. The resulting models show better side chain apolar/polar surface ratio and estimated solvation free energy than structures refined "in vacuo." The consistent lower values of residual NMR constraint violations, apolar/polar surface ratio, and solvation free energy for one of these refined structures allowed prediction of the 3D folding and disulfide connectivity of EETI-II. Except for the few first residues for which no NMR constraints were available, this computer model fully agreed with X-ray structures of CMTI-I (Bode et al.: FEBS Lett. 242:285-292, 1989) and EETI-II complexed with trypsin that appeared after the RMD simulation was completed. Restrained molecular dynamics in water is thus proved to be highly valuable for refinement of DG structures. Also, the successful use of apolar/polar surface ratio and of solvation free energy reinforce the analysis of Novotny et al. (Proteins 4:19-30, 1988) and shows that these criteria are useful indicators of correct versus misfolded models.

Hydrophobic cluster analysis: an efficient new way to compare and analyse amino acid sequences.

A new method for comparing and aligning protein sequences is described. This method, hydrophobic cluster analysis (HCA), relies upon a two-dimensional (2D) representation of the sequences. Hydrophobic clusters are determined in this 2D pattern and then used for the sequence comparisons. The method does not require powerful computer resources and can deal with distantly related proteins, even if no 3D data are available. This is illustrated in the present report by a comparison of human haemoglobin with leghaemoglobin, a comparison of the two domains of liver rhodanese (thiosulphate sulphurtransferase) and a comparison of plastocyanin and azurin.