A Dimerization Site at SCR-17/18 in Factor H Clarifies a New Mechanism for Complement Regulatory Control.

Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20.

Electrostatic interactions modulate the differential aggregation propensities of IgG1 and IgG4P antibodies and inform charged residue substitutions for improved developability.

Native state aggregation is an important concern in the development of therapeutic antibodies. Enhanced knowledge of mAb native state aggregation mechanisms would permit sequence-based selection and design of therapeutic mAbs with improved developability. We investigated how electrostatic interactions affect the native state aggregation of seven human IgG1 and IgG4P mAb isotype pairs, each pair having identical variable domains that are different for each set of IgG1 and IgG4P constructs.

Epitope determines efficacy of therapeutic anti-Tau antibodies in a functional assay with human Alzheimer Tau.

In Alzheimer's disease (AD) and other tauopathies, the cytosolic protein Tau misfolds and forms intracellular aggregates which accumulate within the brain leading to neurodegeneration. Clinical progression is tightly linked to the progressive spread of Tau pathology throughout the brain, and several lines of evidence suggest that Tau aggregates or "seeds" may propagate pathology by spreading from cell to cell in a "prion like" manner. Accordingly, blocking the spread of extracellular seeds with an antibody could be a viable therapeutic approach.

Two distinct conformations of factor H regulate discrete complement-binding functions in the fluid phase and at cell surfaces.

Factor H (FH) is the major regulator of C3b in the alternative pathway of the complement system in immunity. FH comprises 20 short complement regulator (SCR) domains, including eight glycans, and its Y402H polymorphism predisposes those who carry it to age-related macular degeneration. To better understand FH complement binding and self-association, we have studied the solution structures of both the His-402 and Tyr-402 FH allotypes.

Non-linearity of the collagen triple helix in solution and implications for collagen function.

Collagen adopts a characteristic supercoiled triple helical conformation which requires a repeating (Xaa-Yaa-Gly) sequence. Despite the abundance of collagen, a combined experimental and atomistic modelling approach has not so far quantitated the degree of flexibility seen experimentally in the solution structures of collagen triple helices. To address this question, we report an experimental study on the flexibility of varying lengths of collagen triple helical peptides, composed of six, eight, ten and twelve repeats of the most stable Pro-Hyp-Gly (POG) units.

Flexibility in Mannan-Binding Lectin-Associated Serine Proteases-1 and -2 Provides Insight on Lectin Pathway Activation.

The lectin pathway of complement is activated by complexes comprising a recognition component (mannose-binding lectin, serum ficolins, collectin-LK or collectin-K1) and a serine protease (MASP-1 or MASP-2). MASP-1 activates MASP-2, and MASP-2 cleaves C4 and C4b-bound C2. To clarify activation, new crystal structures of Ca-bound MASP dimers were determined, together with their solution structures from X-ray scattering, analytical ultracentrifugation, and atomistic modeling.

A revised mechanism for the activation of complement C3 to C3b: a molecular explanation of a disease-associated polymorphism.

The solution structure of complement C3b is crucial for the understanding of complement activation and regulation. C3b is generated by the removal of C3a from C3. Hydrolysis of the C3 thioester produces C3u, an analog of C3b.

Zinc-induced self-association of complement C3b and Factor H: implications for inflammation and age-related macular degeneration.

The sub-retinal pigment epithelial deposits that are a hallmark of age-related macular degeneration contain both C3b and millimolar levels of zinc. C3 is the central protein of complement, whereas C3u is formed by the spontaneous hydrolysis of the thioester bridge in C3. During activation, C3 is cleaved to form active C3b, then C3b is inactivated by Factor I and Factor H to form the C3c and C3d fragments.

Structure of the discoidin domain receptor 1 extracellular region bound to an inhibitory Fab fragment reveals features important for signaling.

The discoidin domain receptors, DDR1 and DDR2, are constitutively dimeric receptor tyrosine kinases that are activated by triple-helical collagen. Aberrant DDR signaling contributes to several human pathologies, including many cancers. We have generated monoclonal antibodies (mAbs) that inhibit DDR1 signaling without interfering with collagen binding.

Bivalent and co-operative binding of complement factor H to heparan sulfate and heparin.

FH (Factor H) with 20 SCR (short complement regulator) domains is a major serum regulator of complement, and genetic defects in this are associated with inflammatory diseases. Heparan sulfate is a cell-surface glycosaminoglycan composed of sulfated S-domains and unsulfated NA-domains. To elucidate the molecular mechanism of binding of FH to glycosaminoglycans, we performed ultracentrifugation, X-ray scattering and surface plasmon resonance with FH and glycosaminoglycan fragments.

Complement factor H-ligand interactions: self-association, multivalency and dissociation constants.

Factor H (FH) is the major plasma regulator of the central complement protein C3b in the alternative pathway of complement activation. The elucidation of the FH interactions with five major ligands (below) is complicated by their weak μM dissociation constants K(D) and FH multivalency. We present the first survey of all the K(D) values for the major FH-ligand interactions and critically review their physiological significance.

Zinc binding to the Tyr402 and His402 allotypes of complement factor H: possible implications for age-related macular degeneration.

The Tyr402His polymorphism of complement factor H (FH) with 20 short complement regulator (SCR) domains is associated with age-related macular degeneration (AMD). How FH contributes to disease pathology is not clear. Both FH and high concentrations of zinc are found in drusen deposits, the key feature of AMD.

Analytical ultracentrifugation combined with X-ray and neutron scattering: Experiment and modelling.

Analytical ultracentrifugation and solution scattering provide different multi-parameter structural and compositional information on proteins. The joint application of the two methods supplements high resolution structural studies by crystallography and NMR. We summarise the procedures required to obtain equivalent ultracentrifugation and X-ray and neutron scattering data.

Multiple interactions of complement Factor H with its ligands in solution: a progress report.

Factor H (FH) is the major regulator of the central complement protein C3b in the alternative pathway of complement activation, and is comprised of 20 SCR domains. A FH Tyr402His polymorphism in SCR-7 is associated with age-related macular degeneration (AMD) and leads to deposition of complement in drusen. The unravelling of how FH interacts with five major physiological and patho-physiological ligands is complicated by the weak nature of these interactions, coupled with the multivalency of FH.

Unravelling protein-protein interactions between complement factor H and C-reactive protein using a multidisciplinary strategy.

Experimental studies of protein-protein interactions are very much affected by whether the complexes are fully formed (strong, with nanomolar dissociation constants) or partially dissociated (weak, with micromolar dissociation constants). The functions of the complement proteins of innate immunity are governed by the weak interactions between the activated proteins and their regulators. Complement is effective in attacking pathogens, but not the human host, and imbalances in this process can lead to disease conditions.

Complement factor H binds at two independent sites to C-reactive protein in acute phase concentrations.

Factor H (FH) regulates the activation of C3b in the alternative complement pathway, both in serum and at host cell surfaces. It is composed of 20 short complement regulator (SCR) domains. The Y402H polymorphism in FH is a risk factor for age-related macular degeneration.

Constrained solution scattering modelling of human antibodies and complement proteins reveals novel biological insights.

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.

Electrostatic interactions contribute to the folded-back conformation of wild type human factor H.

Factor H (FH), a major serum regulator of C3b in the complement alternative pathway, is composed of 20 short complement regulator (SCR) domains. Earlier solution structures for FH showed that this has a folded-back domain arrangement and exists as oligomers. To clarify the molecular basis for this, analytical ultracentrifugation and X-ray scattering studies of native FH were performed as a function of NaCl concentration and pH.

Multimeric interactions between complement factor H and its C3d ligand provide new insight on complement regulation.

Activation of C3 to C3b signals the start of the alternative complement pathway. The C-terminal short complement regulator (SCR)-20 domain of factor H (FH), the major serum regulator of C3b, possesses a binding site for C3d, a 35-kDa physiological fragment of C3b. Size distribution analyses of mixtures of SCR-16/20 or FH with C3d by analytical ultracentrifugation in 50 and 137 mM NaCl buffer revealed a range of discrete peaks, showing that multimeric complexes had formed at physiologically relevant concentrations.

Uncontrolled zinc- and copper-induced oligomerisation of the human complement regulator factor H and its possible implications for function and disease.

Polymorphisms in factor H (FH), a major regulator of complement activation, and the accumulation of high zinc concentrations in the outer retina are both associated with age-related macular degeneration. FH is inhibited by zinc, which causes FH to aggregate. To investigate this, we quantitatively studied zinc-induced FH self-association by X-ray scattering and analytical ultracentrifugation to demonstrate uncontrolled FH oligomerisation in conditions corresponding to physiological levels of FH and pathological levels of zinc in the outer retina.

Implications of the progressive self-association of wild-type human factor H for complement regulation and disease.

Factor H (FH) is a major regulator of complement alternative pathway activation. It is composed of 20 short complement regulator (SCR) domains and is genetically associated as a risk factor for age-related macular degeneration. Previous studies on FH suggested that it existed in monomeric and dimeric forms.