Mara Guariento

Structural basis for engagement by complement factor H of C3b on a self surface.

Complement factor H (FH) attenuates C3b molecules tethered by their thioester domains to self surfaces and thereby protects host tissues. Factor H is a cofactor for initial C3b proteolysis that ultimately yields a surface-attached fragment (C3d) corresponding to the thioester domain. We used NMR and X-ray crystallography to study the C3d–FH19–20 complex in atomic detail and identify glycosaminoglycan-binding residues in factor H module 20 of the C3d–FH19–20 complex. Mutagenesis justified the merging of the C3d–FH19–20 structure with an existing C3b–FH1–4 crystal structure. We concatenated the merged structure with the available FH6–8 crystal structure and new SAXS-derived FH1–4, FH8–15 and FH15–19 envelopes. The combined data are consistent with a bent-back factor H molecule that binds through its termini to two sites on one C3b molecule and simultaneously to adjacent polyanionic host-surface markers.

Plasmodium falciparum uses a key functional site in complement receptor type-1 for invasion of human erythrocytes

The Plasmodium falciparum adhesin PfRh4 binds to complement receptor type-1 (CR1) on human erythrocytes and mediates a glycophorin-independent invasion pathway. CR1 is a complement regulator and immune-adherence receptor on erythrocytes required for shuttling of C3b/C4b-opsonized particles to liver and spleen for phagocytosis. Using recombinant CR1 constructs, we mapped the recognition site for PfRh4 to complement control protein modules 1 to 3 (CCP1-3) at the membrane-distal amino terminus of CR1. This region of CR1 binds to C4b and C3b and accelerates decay of both classic pathway and alternative pathway C3 and C5 convertases. CCP1-3 competed for PfRh4 binding to erythroid CR1 and inhibited the PfRh4-CR1 invasion pathways across a wide range of P falciparum strains. PfRh4 did not bind significantly to other CR1 constructs, including CCP15-17, which is 85% identical to CCP1-3. PfRh4 binding to CR1 did not affect its C3b/C4b binding capability, and we show evidence for a ternary complex between CCP1-3, C4b, and PfRh4. PfRh4 binding specifically inhibited CR1s convertase decay-accelerating activity, whereas there was no effect on factor H-mediated decay-accelerating activity. These results increase our understanding of the functional implications of CR1 engagement with PfRh4 and highlight the interplay between complement regulation and infection.

The Central Portion of Factor H (Modules 10-15) Is Compact and Contains a Structurally Deviant CCP Module

The first eight and the last two of 20 complement control protein (CCP) modules within complement factor H (fH) encompass binding sites for C3b and polyanionic carbohydrates. These binding sites cooperate self-surface selectively to prevent C3b amplification, thus minimising complement-mediated damage to host. Intervening fH CCPs, apparently devoid of such recognition sites, are proposed to play a structural role. One suggestion is that the generally small CCPs 10–15, connected by longer-than-average linkers, act as a flexible tether between the two functional ends of fH; another is that the long linkers induce a 180° bend in the middle of fH. To test these hypotheses, we determined the NMR-derived structure of fH12–13 consisting of module 12, shown here to have an archetypal CCP structure, and module 13, which is uniquely short and features a laterally protruding helix-like insertion that contributes to a prominent electropositive patch. The unusually long fH12–13 linker is not flexible. It packs between the two CCPs that are not folded back on each other but form a shallow vee shape; analytical ultracentrifugation and X-ray scattering supported this finding. These two techniques additionally indicate that flanking modules (within fH11–14 and fH10–15) are at least as rigid and tilted relative to neighbours as are CCPs 12 and 13 with respect to one another. Tilts between successive modules are not unidirectional; their principal axes trace a zigzag path. In one of two arrangements for CCPs 10–15 that fit well with scattering data, CCP 14 is folded back onto CCP 13. In conclusion, fH10–15 forms neither a flexible tether nor a smooth bend. Rather, it is compact and has embedded within it a CCP module (CCP 13) that appears to be highly specialised given both its deviant structure and its striking surface charge distribution. A passive, purely structural role for this central portion of fH is unlikely.

Structural Analysis of the C-Terminal Region (Modules 18-20) of Complement Regulator Factor H (FH)

Factor H (FH) is a soluble regulator of the human complement system affording protection to host tissues. It selectively inhibits amplification of C3b, the activation-specific fragment of the abundant complement component C3, in fluid phase and on self-surfaces and accelerates the decay of the alternative pathway C3 convertase, C3bBb. We have determined the crystal structure of the three carboxyl-terminal complement control protein (CCP) modules of FH (FH18–20) that bind to C3b, and which additionally recognize polyanionic markers specific to self-surfaces. These CCPs harbour nearly 30 disease-linked missense mutations. We have also deployed small-angle X-ray scattering (SAXS) to investigate FH18–20 flexibility in solution using FH18–20 and FH19–20 constructs. In the crystal lattice FH18–20 adopts a “J”-shape: A ∼122-degree tilt between the structurally highly similar modules 18 and 19 precedes an extended, linear arrangement of modules 19 and 20 as observed in previously determined structures of these two modules alone. However, under solution conditions FH18–20 adopts multiple conformations mediated by flexibility between CCPs 18 and 19. We also pinpoint the locations of disease-associated missense mutations on the module 18 surface and discuss our data in the context of the C3b:FH interaction.

A single amino acid mutation in zebrafish (Danio rerio) liver bile acid-binding protein can change the stoichiometry of ligand binding.

In all of the liver bile acid-binding proteins (L-BABPs) studied so far, it has been found that the stoichiometry of binding is of two cholate molecules per internal binding site. In this paper, we describe the expression, purification, crystallization, and three-dimensional structure determination of zebrafish (Danio rerio) L-BABP to 1.5Å resolution, which is currently the highest available for a protein of this family. Since we have found that in zebrafish, the stoichiometry of binding in the protein cavity is of only one cholate molecule per wild type L-BABP, we examined the role of two crucial amino acids present in the binding site. Using site-directed mutagenesis, we have prepared, crystallized, and determined the three-dimensional structure of co-crystals of two mutants. The mutant G55R has the same stoichiometry of binding as the wild type protein, whereas the C91T mutant changes the stoichiometry of binding from one to two ligand molecules in the cavity and therefore appears to be more similar to the other members of the L-BABP family. Based on the presence or absence of a single disulfide bridge, it can be postulated that fish should bind a single cholate molecule, whereas amphibians and higher vertebrates should bind two. Isothermal titration calorimetry has also revealed the presence in the wild type protein and the G55R mutant of an additional binding site, different from the first and probably located on the surface of the molecule.

Using Mutagenesis and Structural Biology to Map the Binding Site for the Plasmodium falciparum Merozoite Protein PfRh4 on the Human Immune Adherence Receptor

Background: Plasmodium falciparum merozoites invade erythrocytes via interaction of a pathogen protein PfRh4 with a host membrane receptor. Results: The PfRh4-binding site on the human erythrocyte host receptor was mapped by mutagenesis and structural methodology. Conclusion: PfRh4 binds at the receptor terminus in a region overlapping a regulatory functional site. Significance: Understanding the molecular basis of erythrocyte invasion will aid in design of therapeutics. To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1–3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1–3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an ∼30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.

Chicken ileal bile-acid-binding protein: a promising target of investigation to understand binding co-operativity across the protein family

Protein-bile acid interactions are crucial microscopic events at the basis of both physiological and pathological biochemical pathways. BABPs (bile-acid-binding proteins) are intracellular transporters able to bind ligands with different stoichiometry, selectivity and co-operativity. The molecular determinants and energetics of interaction are the observables that connect the microscopic to the macroscopic frameworks. The present paper addresses the study and proposes a mechanism for the multi-site interaction of bile acids with chicken I-BABP (ileal BABP) with the aim of elucidating the determinants of ligand binding in comparison with homologous proteins from different species and tissues. A thermodynamic binding model describing two independent consecutive binding sites is derived from isothermal titration calorimetry experiments and validated on the basis of both protein-observed and ligand-observed NMR titration data. It emerges that a singly bound protein is relatively abundant at low ligand/protein molar ratios assessing the absence of strong co-operativity. Both the measured energetics of binding and the distributed protein chemical-shift perturbations are in agreement with a first binding event triggering a global structural rearrangement. The enthalpic and entropic contributions associated with binding of the first ligand indicate that the interaction increases stability and order of the bound protein. The results described in the present study point to the presence of a protein scaffold which is able to establish long-range communication networks, but does not manifest positive-binding co-operativity, as observed for the human protein. We consider chicken I-BABP a suitable model to address the molecular basis for a gain-of-function on going from non-mammalian to mammalian species.