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Phospholipase A2s hydrolyze aggregated phospholipid substrates much more rapidly than dispersed monomeric ones. Whether this is a consequence of interface-associated conformational changes of the enzyme or of the substrate, or of both, remains a key question in lipid enzymology. This problem is addressed herein using a rationally designed probe of substrate conformation. (1,3/2)-1-O-(phosphorylcholine)-2,3-O-dihexanoylcyclopentane-1,2,3 -triol is a novel short chain phosphatidylcholine analogue in which the glycerol-like backbone is part of a five-membered ring and therefore covalently constrained within a small defined range of conformations. To the extent that the constrained analogue resists aggregation-associated conformational changes, it provides a means for assessing the contribution of such changes to phospholipase A2 action on aggregated phospholipids. The monomeric (-)-cyclopentanoid analogue is a substrate for phospholipase A2s from Naja naja naja venom. However, when this constrained phospholipid is aggregated, its hydrolysis rate is not enhanced, in contrast to its unconstrained counterpart, 1,2-dihexanoyl-sn-glycero-3- phosphorylcholine. This lack of activation was not caused by a failure of the enzyme to bind the micellar, constrained analogue. While the constrained analogue does not show interfacial activation, it does show the activation of phosphatidylethanolamine hydrolysis typical of phosphorylcholine-containing lipids. Hence, these results strongly support the contention that specific packing-induced conformations of aggregated substrate play a substantial role in the large interfacial activations observed with phospholipase A2.

Background: Komagataella phaffii (Pichia pastoris) is a methylotrophic commercially important non-conventional species of yeast that grows in a fermentor to exceptionally high densities on simple media and secretes recombinant proteins efficiently. Genetic engineering strategies are being explored in this organism to facilitate cost-effective biomanufacturing. Small, stable artificial chromosomes in K. phaffii could offer unique advantages by accommodating multiple integrations of extraneous genes and their promoters without accumulating perturbations of native chromosomes or exhausting the availability of selection markers., Results: Here, we describe a linear "nano"chromosome (of 15-25 kb) that, according to whole-genome sequencing, persists in K. phaffii over many generations with a copy number per cell of one, provided non-homologous end joining is compromised (by KU70-knockout). The nanochromosome includes a copy of the centromere from K. phaffii chromosome 3, a K. phaffii-derived autonomously replicating sequence on either side of the centromere, and a pair of K. phaffii-like telomeres. It contains, within its q arm, a landing zone in which genes of interest alternate with long (approx. 1-kb) non-coding DNA chosen to facilitate homologous recombination and serve as spacers. The landing zone can be extended along the nanochromosome, in an inch-worming mode of sequential gene integrations, accompanied by recycling of just two antibiotic-resistance markers. The nanochromosome was used to express PDI, a gene encoding protein disulfide isomerase. Co-expression with PDI allowed the production, from a genomically integrated gene, of secreted murine complement factor H, a plasma protein containing 40 disulfide bonds. As further proof-of-principle, we co-expressed, from a nanochromosome, both PDI and a gene for GFP-tagged human complement factor H under the control of P AOX1 and demonstrated that the secreted protein was active as a regulator of the complement system., Conclusions: We have added K. phaffii to the list of organisms that can produce human proteins from genes carried on a stable, linear, artificial chromosome. We envisage using nanochromosomes as repositories for numerous extraneous genes, allowing intensive engineering of K. phaffii without compromising its genome or weakening the resulting strain., (© 2023. The Author(s).)

Purpose: Factor H (FH, encoded by CFH) prevents activation of the complement system's alternative pathway (AP) on host tissues. FH impedes C3 convertase (C3bBb) formation, accelerates C3bBb decay, and is a cofactor for factor I (FI)-catalyzed C3b cleavage. Numerous CFH variants are associated with age-related macular degeneration (AMD), but their functional consequences frequently remain undetermined. Here, we conduct functional comparisons between a control version of FH (not AMD linked) and 21 AMD-linked FH variants., Methods: Recombinantly produced, untagged, full-length FH versions were assayed for binding to C3b and decay acceleration of C3bBb using surface-plasmon resonance, FI-cofactor activity using a fluorescent probe of C3b integrity, suppression of C5b-9 assembly on an AP-activating surface, and inhibition of human AP-mediated lysis of sheep erythrocytes., Results: All versions were successfully purified despite below-average yields for Arg2Thr, Arg53Cys, Arg175Pro, Arg175Gln, Ile221Val, Tyr402His, Pro503Ala, Arg567Gly, Gly1194Asp, and Arg1210Cys. Compared to control FH, Arg2Thr, Leu3Val, Ser58Ala, Asp90Gly, Asp130Asn, Gln400Lys, Tyr402His, Gly650Val, Ser890Ile, and Thr965Met showed minimal functional differences. Arg1210C, Arg53His, Arg175Gln, Gly1194Asp, Pro503Ala, Arg53Cys, Arg576Gly, and Arg175Pro (in order of decreasing efficacy) underperformed, while Ile221Val, Arg303Gln, and Arg303Trp were "marginal." We newly identified variants toward the center of the molecule, Pro503Ala and Arg567Gly, as potentially pathogenic., Conclusions: Our approach could be extended to other variants of uncertain significance and to assays for noncanonical FH activities, aiming to facilitate selection of cohorts most likely to benefit from therapeutic FH. This is timely as recombinant therapeutic FH is in development for intravitreal treatment of AMD in patients with reduced FH functionality.

Purpose: GEM103 is a recombinantly produced full-length version of the human complement factor H (CFH) under clinical investigation for treatment of age-related macular degeneration (AMD) in individuals carrying an AMD risk-associated genetic variant of CFH . This study aimed to investigate the complement pathway-related functions of GEM103 in comparison with those of native human CFH., Methods: Key biological activities of GEM103 and human serum-derived CFH (sdCFH) were compared using four independent functional assays. Assays of C3b binding and C3 convertase decay-accelerating activity (DAA) were performed by surface plasmon resonance (SPR). Cofactor activity (CA) was measured using 8-anilinonaphthalene-1-sulfonic acid as a fluorescent probe of C3b integrity. The abilities of GEM103 and sdCFH to protect sheep erythrocytes from hemolysis by CFH-depleted normal human serum were assessed colorimetrically., Results: In multiple SPR-based assays of C3b binding and DAA, the performance of GEM103 was consistently comparable to that of sdCFH across a range of matching concentrations. The EC 50 ± SD in the fluorescence-based fluid-phase CA assay was 0.21 ± 0.06 µM for GEM103 compared to 0.20 ± 0.09 µM for sdCFH. In hemolysis assays, the EC 50 value of 0.33 ± 0.16 µM for GEM103 versus 0.46 ± 0.06 µM for sdCFH were not significantly different ( p  = 0.81)., Conclusions: GEM103, a recombinant CFH developed by Gemini Therapeutics, shows activity profiles comparable to sdCFH in all complement-related assays employed in this study, suggesting that GEM103 is equivalent to the native glycoprotein in terms of its in vitro functional activity. These results support further study of GEM103 as a potential therapy for AMD.

Many proteins recognise other proteins via mechanisms that involve the folding of intrinsically disordered regions upon complex formation. Here we investigate how the selectivity of a drug-like small molecule arises from its modulation of a protein disorder-to-order transition. Binding of the compound AM-7209 has been reported to confer order upon an intrinsically disordered 'lid' region of the oncoprotein MDM2. Calorimetric measurements revealed that truncation of the lid region of MDM2 increases the apparent dissociation constant of AM-7209 250-fold. By contrast, lid truncation has little effect on the binding of the ligand Nutlin-3a. Insights into these differential binding energetics were obtained via a complete thermodynamic analysis that featured adaptive absolute alchemical free energy of binding calculations with enhanced-sampling molecular dynamics simulations. The simulations reveal that in apo MDM2 the ordered lid state is energetically disfavoured. AM-7209, but not Nutlin-3a, shows a significant energetic preference for ordered lid conformations, thus shifting the balance towards ordering of the lid in the AM-7209/MDM2 complex. The methodology reported herein should facilitate broader targeting of intrinsically disordered regions in medicinal chemistry., Competing Interests: J. M. is a current member of the Scientific Advisory Board of Cresset., (This journal is © The Royal Society of Chemistry.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Recombinant human factor H (hFH) has potential for treating diseases linked to aberrant complement regulation including C3 glomerulopathy (C3G) and dry age-related macular degeneration. Murine FH (mFH), produced in the same host, is useful for pre-clinical investigations in mouse models of disease. An abundance of FH in plasma suggests high doses, and hence microbial production, will be needed. Previously, Pichia pastoris produced useful but modest quantities of hFH. Herein, a similar strategy yielded miniscule quantities of mFH. Since FH has 40 disulfide bonds, we created a P. pastoris strain containing a methanol-inducible codon-modified gene for protein-disulfide isomerase (PDI) and transformed this with codon-modified DNA encoding mFH under the same promoter. What had been barely detectable yields of mFH became multiple 10s of mg/L. Our PDI-overexpressing strain also boosted hFH overproduction, by about tenfold. These enhancements exceeded PDI-related production gains reported for other proteins, all of which contain fewer disulfide-stabilized domains. We optimized fermentation conditions, purified recombinant mFH, enzymatically trimmed down its (non-human) N-glycans, characterised its functions in vitro and administered it to mice. In FH-knockout mice, our de-glycosylated recombinant mFH had a shorter half-life and induced more anti-mFH antibodies than mouse serum-derived, natively glycosylated, mFH. Even sequential daily injections of recombinant mFH failed to restore wild-type levels of FH and C3 in mouse plasma beyond 24 hours after the first injection. Nevertheless, mFH functionality appeared to persist in the glomerular basement membrane because C3-fragment deposition here, a hallmark of C3G, remained significantly reduced throughout and beyond the ten-day dosing regimen., Competing Interests: KM has received consultancy or research income from Gemini Therapeutics Inc, Freeline Therapeutics, MPM Capital, Idorsia Pharmaceuticals Ltd and Catalyst Biosciences. PB is a scientific co-founder of, and has received consultancy and research income from, Gemini Therapeutics Inc. AH is founder and Chief Scientific Officer of Invizius. EM is currently an employee of Invizius. AR has been employed at GlaxoSmithKline since October 2014. Results reported in this work were undertaken during her Wellcome Trust Intermediate Clinical Fellowship. Her spouse, David Kavanagh, is head of the National Renal Complement Therapeutics Centre, UK, and a board member and scientific advisor to Gyroscope Therapeutics Ltd. PNB and AR are co-authors on a recombinant CFH patent application (EP10803266A). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kerr, Herbert, Makou, Abramczyk, Malik, Lomax-Browne, Yang, Pappworth, Denton, Richards, Marchbank, Pickering and Barlow.)

Membranoproliferative glomerulonephritis (MPGN), C3 glomerulopathy (C3G), atypical haemolytic uraemic syndrome (aHUS) and age-related macular degeneration (AMD) have all been strongly linked with dysfunction of the alternative pathway (AP) of complement. A significant proportion of individuals with MPGN, C3G, aHUS and AMD carry rare genetic variants in the CFH gene that cause functional or quantitative deficiencies in the factor H (FH) protein, an important regulator of the AP. In silico analysis of the deleteriousness of rare genetic variants in CFH is not reliable and careful biochemical assessment remains the gold standard. Six N-terminal variants of uncertain significance in CFH were identified in patients with these diseases of the AP and selected for analysis. The variants were produced in Pichia Pastoris in the setting of FH CCPs 1-4, purified by nickel affinity chromatography and size exclusion and characterized by surface plasmon resonance and haemolytic assays as well as by cofactor assays in the fluid phase. A single variant, Q81P demonstrated a profound loss of binding to C3b with consequent loss of cofactor and decay accelerating activity. A further 2 variants, G69E and D130N, demonstrated only subtle defects which could conceivably over time lead to disease progression of more chronic AP diseases such as C3G and AMD. In the variants S159N, A161S, and M162V any functional defect was below the capacity of the experimental assays to reliably detect. This study further underlines the importance of careful biochemical assessment when assigning functional consequences to rare genetic variants that may alter clinical decisions for patients., Competing Interests: EKSW has received consultancy income from Alexion Pharmaceuticals, Biocryst, and Novartis. KJM, has received consultancy income from Gemini Therapeutics Freeline Therapeutics, MPM Capital, Catalyst Biosciences. CLH has received consultancy income from Roche, GSK, Gyroscope Therapeutics, Q32 Bio, Freeline Therapeutics, Ra Pharmaceuticals, and Biocryst. DK has received consultancy income from Gyroscope Therapeutics, Alexion Pharmaceuticals, Novartis, Apellis and Sarepta. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wong, Hallam, Brocklebank, Walsh, Smith-Jackson, Shuttleworth, Cox, Anderson, Barlow, Marchbank, Harris and Kavanagh.)

Macrophages are a major immune cell type in the tumor microenvironment, where they display a tumor-supporting phenotype. Factor H (FH) is a complement inhibitor that also plays a role in several cellular functions. To date, the phenotype of monocytes stimulated with FH has been unexplored. We discovered that FH is a survival factor for CD14 + primary human monocytes, promoting their differentiation into macrophages in serum-free medium. This activity was localized to the C-terminal domains of FH and it was inhibited in plasma, indicating that the phenomenon may be most relevant in tissues. FH-induced macrophages display characteristics of immunosuppressive cells including expression of CD163 and CD206, release of the anti-inflammatory cytokine IL-10 and changes in metabolism. Furthermore, FH-induced macrophages express low levels of HLA-DR but high levels of co-inhibitory molecule programmed death-ligand 1 (PD-L1), and accordingly, a reduced capacity for T-cell activation. Finally, we show that FH is expressed by human breast cancer cells and that this correlates with the presence of immunosuppressive macrophages, breast cancer recurrence and severity of the disease. We propose that the expression of FH by tumor cells and the promotion of an immunosuppressive cancer microenvironment by this protein should be taken into account when considering the effectiveness of immunotherapies against breast cancer., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Proteins need to interconvert between many conformations in order to function, many of which are formed transiently, and sparsely populated. Particularly when the lifetimes of these states approach the millisecond timescale, identifying the relevant structures and the mechanism by which they interconvert remains a tremendous challenge. Here we introduce a novel combination of accelerated MD (aMD) simulations and Markov state modelling (MSM) to explore these 'excited' conformational states. Applying this to the highly dynamic protein CypA, a protein involved in immune response and associated with HIV infection, we identify five principally populated conformational states and the atomistic mechanism by which they interconvert. A rational design strategy predicted that the mutant D66A should stabilise the minor conformations and substantially alter the dynamics, whereas the similar mutant H70A should leave the landscape broadly unchanged. These predictions are confirmed using CPMG and R 1ρ solution state NMR measurements. By efficiently exploring functionally relevant, but sparsely populated conformations with millisecond lifetimes in silico , our aMD/MSM method has tremendous promise for the design of dynamic protein free energy landscapes for both protein engineering and drug discovery., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Activation and suppression of the complement system compete on every serum-exposed surface, host or foreign. Potentially harmful outcomes of this competition depend on surface molecules through mechanisms that remain incompletely understood. Combining surface plasmon resonance (SPR) with atomic force microscopy (AFM), here we studied two complement system proteins at the single-molecule level: C3b, the proteolytically activated form of C3, and factor H (FH), the surface-sensing C3b-binding complement regulator. We used SPR to monitor complement initiation occurring through a positive-feedback loop wherein surface-deposited C3b participates in convertases that cleave C3, thereby depositing more C3b. Over multiple cycles of flowing factor B, factor D, and C3 over the SPR chip, we amplified C3b from ∼20 to ∼220 molecules·μm -2 AFM revealed C3b clusters of up to 20 molecules and solitary C3b molecules deposited up to 200 nm away from the clusters. A force of 0.17 ± 0.02 nanonewtons was needed to pull a single FH molecule, anchored to the AFM probe, from its complex with surface-attached C3b. The extent to which FH molecules stretched before detachment varied widely among complexes. Performing force-distance measurements with FH(D1119G), a variant lacking one of the C3b-binding sites and causing atypical hemolytic uremic syndrome, we found that it detached more uniformly and easily. In further SPR experiments, K D values between FH and C3b on a custom-made chip surface were 5-fold tighter than on commercial chips and similar to those on erythrocytes. These results suggest that the chemistry at the surface on which FH acts drives conformational adjustments that are functionally critical., (© 2019 Makou et al.)

An effective vaccine is a priority for malaria control and elimination. The leading candidate in the Plasmodium falciparum blood stage is PfRh5. PfRh5 assembles into trimeric complex with PfRipr and PfCyRPA in the parasite, and this complex is essential for erythrocyte invasion. In this study, we show that antibodies specific for PfRh5 and PfCyRPA prevent trimeric complex formation. We identify the EGF-7 domain on PfRipr as a neutralising epitope and demonstrate that antibodies against this region act downstream of complex formation to prevent merozoite invasion. Antibodies against the C-terminal region of PfRipr were more inhibitory than those against either PfRh5 or PfCyRPA alone, and a combination of antibodies against PfCyRPA and PfRipr acted synergistically to reduce invasion. This study supports prioritisation of PfRipr for development as part of a next-generation antimalarial vaccine., (© 2019 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

Atypical hemolytic uremic syndrome (aHUS) is frequently associated in humans with loss-of-function mutations in complement-regulating proteins or gain-of-function mutations in complement-activating proteins. Thus, aHUS provides an archetypal complement-mediated disease with which to model new therapeutic strategies and treatments. Herein, we show that, when transferred to mice, an aHUS-associated gain-of-function change (D1115N) to the complement-activation protein C3 results in aHUS. Homozygous C3 p.D1115N (C3KI) mice developed spontaneous chronic thrombotic microangiopathy together with hematuria, thrombocytopenia, elevated creatinine, and evidence of hemolysis. Mice with active disease had reduced plasma C3 with C3 fragment and C9 deposition within the kidney. Therapeutic blockade or genetic deletion of C5, a protein downstream of C3 in the complement cascade, protected homozygous C3KI mice from thrombotic microangiopathy and aHUS. Thus, our data provide in vivo modeling evidence that gain-of-function changes in complement C3 drive aHUS. They also show that long-term C5 deficiency is not accompanied by development of other renal complications (such as C3 glomerulopathy) despite sustained dysregulation of C3. Our results suggest that this preclinical model will allow testing of novel complement inhibitors with the aim of developing precisely targeted therapeutics that could have application in many complement-mediated diseases.

Cyclophilins (Cyps) are a major family of drug targets that are challenging to prosecute with small molecules because the shallow nature and high degree of conservation of the active site across human isoforms offers limited opportunities for potent and selective inhibition. Herein a computational approach based on molecular dynamics simulations and free energy calculations was combined with biophysical assays and X-ray crystallography to explore a flip in the binding mode of a reported urea-based Cyp inhibitor. This approach enabled access to a distal pocket that is poorly conserved among key Cyp isoforms, and led to the discovery of a new family of sub-micromolar cell-active inhibitors that offer unprecedented opportunities for the development of next-generation drug therapies based on Cyp inhibition. The computational approach is applicable to a broad range of organic functional groups and could prove widely enabling in molecular design.

Background C3 glomerulopathy (C3G) is associated with dysregulation of the alternative pathway of complement activation, and treatment options for C3G remain limited. Complement factor H (FH) is a potent regulator of the alternative pathway and might offer a solution, but the mass and complexity of FH makes generation of full-length FH far from trivial. We previously generated a mini-FH construct, with FH short consensus repeats 1-5 linked to repeats 18-20 (FH 1-5^18-20 ), that was effective in experimental C3G. However, the serum t 1/2 of FH 1-5^18-20 was significantly shorter than that of serum-purified FH. Methods We introduced the oligomerization domain of human FH-related protein 1 (denoted by R1-2) at the carboxy or amino terminus of human FH 1-5^18-20 to generate two homodimeric mini-FH constructs (FH R1-2^1-5^18-20 and FH 1-5^18-20^R1-2 , respectively) in Chinese hamster ovary cells and tested these constructs using binding, fluid-phase, and erythrocyte lysis assays, followed by experiments in FH-deficient Cfh-/- mice. Results FH R1-2^1-5^18-20 and FH 1-5^18-20^R1-2 homodimerized in solution and displayed avid binding profiles on clustered C3b surfaces, particularly FH R1-2^1-5^18-20 Each construct was >10-fold more effective than FH at inhibiting cell surface complement activity in vitro and restricted glomerular basement membrane C3 deposition in vivo significantly better than FH or FH 1-5^18-20 FH 1-5^18-20^R1-2 had a C3 breakdown fragment binding profile similar to that of FH, a >5-fold increase in serum t 1/2 compared with that of FH 1-5^18-20 , and significantly better retention in the kidney than FH or FH 1-5^18-20 Conclusions FH 1-5^18-20^R1-2 may have utility as a treatment option for C3G or other complement-mediated diseases., (Copyright © 2018 by the American Society of Nephrology.)

Spontaneous activation enables the complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 complement-control protein modules (CCPs 1-20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in Pichia pastoris We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes ( E S ) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on E S but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing complement-mediated hemolysis of E S PspCN, a CFH-binding Streptococcus pneumoniae protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on E S Conversely, PspCN boosted the CA, on E S , of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH variants., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The slow but spontaneous and ubiquitous formation of C3(H2O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H2O) has not been determined. Here we subjected C3(H2O) to quantitative cross-linking/mass spectrometry (QCLMS). This revealed details of the structural differences and similarities between C3(H2O) and C3, as well as between C3(H2O) and its pivotal proteolytic cleavage product, C3b, which shares functionally similarity with C3(H2O). Considered in combination with the crystal structures of C3 and C3b, the QCMLS data suggest that C3(H2O) generation is accompanied by the migration of the thioester-containing domain of C3 from one end of the molecule to the other. This creates a stable C3b-like platform able to bind the zymogen, factor B, or the regulator, factor H. Integration of available crystallographic and QCLMS data allowed the determination of a 3D model of the C3(H2O) domain architecture. The unique arrangement of domains thus observed in C3(H2O), which retains the anaphylatoxin domain (that is excised when C3 is enzymatically activated to C3b), can be used to rationalize observed differences between C3(H2O) and C3b in terms of complement activation and regulation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The human complement system is the frontline defense mechanism against invading pathogens. The coexistence of humans and microbes throughout evolution has produced ingenious molecular mechanisms by which microorganisms escape complement attack. A common evasion strategy used by diverse pathogens is the hijacking of soluble human complement regulators to their surfaces to afford protection from complement activation. One such host regulator is factor H (FH), which acts as a negative regulator of complement to protect host tissues from aberrant complement activation. In this report, we show that Plasmodium falciparum merozoites, the invasive form of the malaria parasites, actively recruit FH and its alternative spliced form FH-like protein 1 when exposed to human serum. We have mapped the binding site in FH that recognizes merozoites and identified Pf92, a member of the six-cysteine family of Plasmodium surface proteins, as its direct interaction partner. When bound to merozoites, FH retains cofactor activity, a key function that allows it to downregulate the alternative pathway of complement. In P. falciparum parasites that lack Pf92, we observed changes in the pattern of C3b cleavage that are consistent with decreased regulation of complement activation. These results also show that recruitment of FH affords P. falciparum merozoites protection from complement-mediated lysis. Our study provides new insights on mechanisms of immune evasion of malaria parasites and highlights the important function of surface coat proteins in the interplay between complement regulation and successful infection of the host., (Copyright © 2016 by The American Association of Immunologists, Inc.)

The serum proteins factor H (FH), consisting of 20 complement control protein modules (CCPs), and its splice product FH-like protein 1 (FHL-1; consisting of CCPs 1-7) are major regulators of the alternative pathway (AP) of complement activation. The engineered version of FH, miniFH, contains only the N- and C-terminal portions of FH linked by an optimized peptide and shows ∼ 10-fold higher ex vivo potency. We explored the hypothesis that regulatory potency is enhanced by unmasking of a ligand-binding site in the C-terminal CCPs 19-20 that is cryptic in full-length native FH. Therefore, we produced an FH variant lacking the central domains 10-15 (FHΔ10-15). To explore how avidity affects regulatory strength, we generated a duplicated version of miniFH, termed midiFH. We compared activities of FHΔ10-15 and midiFH to miniFH, FH, and FHL-1. Relative to FH, FHΔ10-15 exhibited an altered binding profile toward C3 activation products and a 5-fold-enhanced complement regulation on a paroxysmal nocturnal hemoglobinuria patient's erythrocytes. Contrary to dogma, FHL-1 and FH exhibited equal regulatory activity, suggesting that the role of FHL-1 in AP regulation has been underestimated. Unexpectedly, a substantially increased avidity for complement opsonins, as seen in midiFH, did not potentiate the inhibitory potential on host cells. In conclusion, comparisons of engineered and native FH-based regulators have identified features that determine high AP regulatory activity on host cells. Unrestricted availability of FH CCPs 19-20 and an optimal spatial orientation between the N- and C-terminal FH regions are key., (Copyright © 2016 by The American Association of Immunologists, Inc.)

In an attempt to evade annihilation by the vertebrate complement system, many microbes capture factor H (FH), the key soluble complement-regulating protein in human plasma. However, FH is normally an active complement suppressor exclusively on self-surfaces and this selective action of FH is pivotal to self versus non-self discrimination by the complement system. We investigated whether the bacterially captured FH becomes functionally enhanced and, if so, how this is achieved at a structural level. We found, using site-directed and truncation mutagenesis, surface plasmon resonance, nuclear magnetic resonance spectroscopy, and cross-linking and mass spectrometry, that the N-terminal domain of Streptococcus pneumoniae protein PspC (PspCN) not only binds FH extraordinarily tightly but also holds it in a previously uncharacterized conformation. Functional enhancement arises from exposure of a C-terminal cryptic second binding site in FH for C3b, the activation-specific fragment of the pivotal complement component, C3. This conformational change of FH doubles its affinity for C3b and increases 5-fold its ability to accelerate decay of the binary enzyme (C3bBb) responsible for converting C3 to C3b in an amplification loop. Despite not sharing critical FH-binding residues, PspCNs from D39 and Tigr4 S. pneumoniae exhibit similar FH-anchoring and enhancing properties. We propose that these bacterial proteins mimic molecular markers of self-surfaces, providing a compelling hypothesis for how FH prevents complement-mediated injury to host tissue while lacking efficacy on virtually all other surfaces. In hemolysis assays with 2-aminoethylisothiouronium bromide-treated erythrocytes that recapitulate paroxysmal nocturnal hemoglobinuria, PspCN enhanced protection of cells by FH, suggesting a new paradigm for therapeutic complement suppression., (Copyright © 2015 The Authors.)

Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a β-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins., (© 2015 Authors; published by Portland Press Limited.)

During blood stage Plasmodium falciparum infection, merozoites invade uninfected erythrocytes via a complex, multistep process involving a series of distinct receptor-ligand binding events. Understanding each element in this process increases the potential to block the parasite's life cycle via drugs or vaccines. To investigate specific receptor-ligand interactions, they were systematically blocked using a combination of genetic deletion, enzymatic receptor cleavage and inhibition of binding via antibodies, peptides and small molecules, and the resulting temporal changes in invasion and morphological effects on erythrocytes were filmed using live cell imaging. Analysis of the videos have shown receptor-ligand interactions occur in the following sequence with the following cellular morphologies; 1) an early heparin-blockable interaction which weakly deforms the erythrocyte, 2) EBA and PfRh ligands which strongly deform the erythrocyte, a process dependant on the merozoite's actin-myosin motor, 3) a PfRh5-basigin binding step which results in a pore or opening between parasite and host through which it appears small molecules and possibly invasion components can flow and 4) an AMA1-RON2 interaction that mediates tight junction formation, which acts as an anchor point for internalization. In addition to enhancing general knowledge of apicomplexan biology, this work provides a rational basis to combine sequentially acting merozoite vaccine candidates in a single multi-receptor-blocking vaccine.

Complement C3 activation is a characteristic finding in membranoproliferative GN (MPGN). This activation can be caused by immune complex deposition or an acquired or inherited defect in complement regulation. Deficiency of complement factor H has long been associated with MPGN. More recently, heterozygous genetic variants have been reported in sporadic cases of MPGN, although their functional significance has not been assessed. We describe a family with MPGN and acquired partial lipodystrophy. Although C3 nephritic factor was shown in family members with acquired partial lipodystrophy, it did not segregate with the renal phenotype. Genetic analysis revealed a novel heterozygous mutation in complement factor H (R83S) in addition to known risk polymorphisms carried by individuals with MPGN. Patients with MPGN had normal levels of factor H, and structural analysis of the mutant revealed only subtle alterations. However, functional analysis revealed profoundly reduced C3b binding, cofactor activity, and decay accelerating activity leading to loss of regulation of the alternative pathway. In summary, this family showed a confluence of common and rare functionally significant genetic risk factors causing disease. Data from our analysis of these factors highlight the role of the alternative pathway of complement in MPGN., (Copyright © 2014 by the American Society of Nephrology.)

In this study, we examine the feasibility and limitations of describing the motional behavior of three-domain proteins in which the domains are linearly connected. In addition to attempting the determination of the internal and overall reorientational correlation times, we investigate the existence of correlations in the motions between the three domains. Since in linearly arranged three-domain proteins, there are typically no experimental data that can directly report on motional correlation between the first and the third domain, we address this question by dynamics simulations. Two limiting cases occur: (1) for weak repulsive potentials and (2) when strong repulsive potentials are applied between sequential domains. The motions of the terminal domains become correlated in the case of strong interdomain repulsive potentials when these potentials do not allow the angle between the sequential domains to be smaller than about 60°. Using the model-free (MF) and extended MF formalisms of Lipari and Szabo, we find that the motional behavior can be separated into two components; the first component represents the concerted overall motion of the three domains, and the second describes the independent component of the motion of each individual domain. We find that this division of the motional behavior of the protein is maintained only when their timescales are distinct and can be made when the angles between sequential domains remain between 60° and 160°. In this work, we identify and quantify interdomain motional correlations., (Copyright © 2013 Wiley Periodicals, Inc.)

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.

Human C7 is one of four homologous complement proteins that self-assemble on the nascent activation-specific fragment, C5b, thus forming the cytolytic membrane attack complex (MAC). In addition to the conserved modular core of the MAC/perforin protein family, C7 has four C-terminal domains comprising a pair of complement control protein modules (CCPs) preceding two Factor-I like modules (FIMs). It is proposed that the C7-CCPs might serve as a molecular arm for delivery of C7-FIMs to their binding site on C5b. Here we present the NMR chemical shift assignments for the C7-CCPs produced as a 14-kDa recombinant protein. Based upon triple-resonance experiments, 98 and 94 % of the backbone and side-chain ((1)H, (13)C and (15)N) assignments, respectively, have been completed. The chemical shifts and assignments have been deposited in the BioMagResBank database under accession number 18530.

Factor H (FH) is a soluble regulator of the proteolytic cascade at the core of the evolutionarily ancient vertebrate complement system. Although FH consists of a single chain of similar protein modules, it has a demanding job description. Its chief role is to prevent complement-mediated injury to healthy host cells and tissues. This entails recognition of molecular patterns on host surfaces combined with control of one of nature's most dangerous examples of a positive-feedback loop. In this way, FH modulates, where and when needed, an amplification process that otherwise exponentially escalates the production of the pro-inflammatory, pro-phagocytic, and pro-cytolytic cleavage products of complement proteins C3 and C5. Mutations and single-nucleotide polymorphisms in the FH gene and autoantibodies against FH predispose individuals to diseases, including age-related macular degeneration, dense-deposit disease, and atypical hemolytic uremic syndrome. Moreover, deletions or variations of genes for FH-related proteins also influence the risk of disease. Numerous pathogens hijack FH and use it for self-defense. As reviewed herein, a molecular understanding of FH function is emerging. While its functional oligomeric status remains uncertain, progress has been achieved in characterizing its three-dimensional architecture and, to a lesser extent, its intermodular flexibility. Models are proposed, based on the reconciliation of older data with a wealth of recent evidence, in which a latent circulating form of FH is activated by its principal target, C3b tethered to a self-surface. Such models suggest hypotheses linking sequence variations to pathophysiology, but improved, more quantitative, functional assays and rigorous data analysis are required to test these ideas.

Therapeutic modulation of the complement system has become increasingly important in line with the growing recognition of the role of complement in numerous diseases. Compstatin, a peptidic inhibitor that acts at the central level of the complement cascade, is currently in clinical evaluation but routes to improve its efficacy have not yet been fully explored. Here, we report improvements in both the inhibitory potency and pharmacokinetic parameters of compstatin that broaden its clinical applications. Selective modification of the compstatin N-terminus with non-proteinogenic amino acids resulted in the first analogue with subnanomolar binding affinity (KD=0.5nM) and other similarly potent derivatives with improved solubility in clinically relevant solvents. Detailed structure-activity relationship studies based on biophysical and computational methods revealed key structural determinants for the observed improvements. Importantly, pharmacokinetic evaluation in non-human primates revealed target-driven elimination kinetics with plasma half-life values exceeding expectations for peptidic drugs (close to 12h). This successful optimization strategy is expected to pave the way for systemic administration of compstatin in a range of clinical conditions., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Factor H (fH) and properdin both modulate complement; however, fH inhibits activation, and properdin promotes activation of the alternative pathway of complement. Mutations in fH associate with several human kidney diseases, but whether inhibiting properdin would be beneficial in these diseases is unknown. Here, we found that either genetic or pharmacological blockade of properdin, which we expected to be therapeutic, converted the mild C3 GN of an fH-mutant mouse to a lethal C3 GN with features of human dense deposit disease. We attributed this phenotypic change to a differential effect of properdin on the dynamics of alternative pathway complement activation in the fluid phase and the cell surface in the fH-mutant mice. Thus, in fH mutation-related C3 glomerulopathy, additional factors that impact the activation of the alternative pathway of complement critically determine the nature and severity of kidney pathology. These results show that therapeutic manipulation of the complement system requires rigorous disease-specific target validation.

The 155-kDa plasma glycoprotein factor H (FH), which consists of 20 complement control protein (CCP) modules, protects self-tissue but not foreign organisms from damage by the complement cascade. Protection is achieved by selective engagement of FH, via CCPs 1-4, CCPs 6-8 and CCPs 19-20, with polyanion-rich host surfaces that bear covalently attached, activation-specific, fragments of complement component C3. The role of intervening CCPs 9-18 in this process is obscured by lack of structural knowledge. We have concatenated new high-resolution solution structures of overlapping recombinant CCP pairs, 10-11 and 11-12, to form a three-dimensional structure of CCPs 10-12 and validated it by small-angle X-ray scattering of the recombinant triple-module fragment. Superimposing CCP 12 of this 10-12 structure with CCP 12 from the previously solved CCP 12-13 structure yielded an S-shaped structure for CCPs 10-13 in which modules are tilted by 80-110° with respect to immediate neighbors, but the bend between CCPs 10 and 11 is counter to the arc traced by CCPs 11-13. Including this four-CCP structure in interpretation of scattering data for the longer recombinant segments, CCPs 10-15 and 8-15, implied flexible attachment of CCPs 8 and 9 to CCP 10 but compact and intimate arrangements of CCP 14 with CCPs 12, 13 and 15. Taken together with difficulties in recombinant production of module pairs 13-14 and 14-15, the aberrant structure of CCP 13 and the variability of 13-14 linker sequences among orthologues, a structural dependency of CCP 14 on its neighbors is suggested; this has implications for the FH mechanism., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Factor H autoantibodies are found in ~10% of aHUS patients. Most are associated with complete deficiency of factor H related proteins 1/3 and bind to the C terminal recognition domain. MPGN, like aHUS, is characterised by complement activation. In this study we, therefore, examined the hypothesis that factor H autoantibodies are associated with MPGN. We screened sera from 16 MPGN patients and 100 normal controls using ELISA and detected strongly positive IgG factor H autoantibodies in 2 patients. One patient had type II (DDD) MPGN (male aged 24 yrs) with C3NeF and the other type I (female aged 26 yrs) with no detectable C3NeF. We identified the binding site of the autoantibodies using small SCR domain fragments in the ELISA and showed that the autoantibodies in both patients bound predominately to the N terminal complement regulatory domain of factor H. We measured CFHR 1/3 copy number using MLPA and showed that both patients had 2 copies of CFHR1 and 3. Finally, we examined the functionality of detected factor H autoantibodies using purified patient IgG and observed increased haemolysis when purified IgG from both patients was added to normal human sera prior to incubation with rabbit red blood cells. Thus, in a cohort of MPGN patients we have found a high titre of functionally significant factor H autoantibodies in two patients with MPGN. Antibody depleting therapy may have a role in such patients and we suggest that screening for factor H autoantibodies should be undertaken in all patients with MPGN., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8-167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly.

Numerous complement factor H (FH) mutations predispose patients to atypical hemolytic uremic syndrome (aHUS) and other disorders arising from inadequately regulated complement activation. No unifying structural or mechanistic consequences have been ascribed to these mutants beyond impaired self-cell protection. The S1191L and V1197A mutations toward the C-terminus of FH, which occur in patients singly or together, arose from gene conversion between CFH encoding FH and CFHR1 encoding FH-related 1. We show that neither single nor double mutations structurally perturbed recombinant proteins consisting of the FH C-terminal modules, 19 and 20 (FH19-20), although all three FH19-20 mutants were poor, compared to wild-type FH19-20, at promoting hemolysis of C3b-coated erythrocytes through competition with full-length FH. Indeed, our new crystal structure of the S1191L mutant of FH19-20 complexed with an activation-specific complement fragment, C3d, was nearly identical to that of the wild-type FH19-20:C3d complex, consistent with mutants binding to C3b with wild-type-like affinity. The S1191L mutation enhanced thermal stability of module 20, whereas the V1197A mutation dramatically decreased it. Thus, although mutant proteins were folded at 37 °C, they differ in conformational rigidity. Neither single substitutions nor double substitutions increased measurably the extent of FH19-20 self-association, nor did these mutations significantly affect the affinity of FH19-20 for three glycosaminoglycans, despite critical roles of module 20 in recognizing polyanionic self-surface markers. Unexpectedly, FH19-20 mutants containing Leu1191 self-associated on a heparin-coated surface to a higher degree than on surfaces coated with dermatan or chondroitin sulfates. Thus, potentially disease-related functional distinctions between mutants, and between FH and FH-related 1, may manifest in the presence of specific glycosaminoglycans.

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.

Complement receptor-type 1 (CR1, CD35) is the immune-adherence receptor, a complement regulator, and an erythroid receptor for Plasmodium falciparum during merozoite invasion and subsequent rosette formation involving parasitized and non-infected erythrocytes. The non-uniform geographical distribution of Knops blood group CR1 alleles Sl1/2 and McC(a/b) may result from selective pressures exerted by differential exposure to infectious hazards. Here, four variant short recombinant versions of CR1 were produced and analyzed, focusing on complement control protein modules (CCPs) 15-25 of its ectodomain. These eleven modules encompass a region (CCPs 15-17) key to rosetting, opsonin recognition and complement regulation, as well as the Knops blood group polymorphisms in CCPs 24-25. All four CR1 15-25 variants were monomeric and had similar axial ratios. Modules 21 and 22, despite their double-length inter-modular linker, did not lie side-by-side so as to stabilize a bent-back architecture that would facilitate cooperation between key functional modules and Knops blood group antigens. Indeed, the four CR1 15-25 variants had virtually indistinguishable affinities for immobilized complement fragments C3b (K(D) = 0.8-1.1 µM) and C4b (K(D) = 5.0-5.3 µM). They were all equally good co-factors for factor I-catalysed cleavage of C3b and C4b, and they bound equally within a narrow affinity range, to immobilized C1q. No differences between the variants were observed in assays for inhibition of erythrocyte invasion by P. falciparum or for rosette disruption. Neither differences in complement-regulatory functionality, nor interactions with P. falciparum proteins tested here, appear to have driven the non-uniform geographic distribution of these alleles.

Structural knowledge of interactions amongst the ~ 40 proteins of the human complement system, which is central to immune surveillance and homeostasis, is expanding due primarily to X-ray diffraction of co-crystallized proteins. Orthogonal evidence, in solution, for the physiological relevance of such co-crystal structures is valuable since intermolecular affinities are generally weak-to-medium and inter-domain mobility may be important. In this current work, Förster resonance energy transfer (FRET) was used to investigate the 10 μM K(D) (210 kD) complex between the N-terminal region of the soluble complement regulator, factor H (FH1-4), and the key activation-specific complement fragment, C3b. Using site-directed mutagenesis, seven cysteines were introduced individually at potentially informative positions within the four CCP modules comprising FH1-4, then used for fluorophore attachment. C3b possesses a thioester domain featuring an internal cycloglutamyl cysteine thioester; upon hydrolysis this yields a free thiol (Cys988) that was also fluorescently tagged. Labeled proteins were functionally active as cofactors for cleavage of C3b to iC3b except for FH1-4(Q40C) where conjugation with the fluorophore likely abrogated interaction with the protease, factor I. Time-resolved FRET measurements were undertaken to explore interactions between FH1-4 and C3b in fluid phase and under near-physiological conditions. These experiments confirmed that, as in the cocrystal structure, FH1-4 binds to C3b with CCP module 1 furthest from, and CCP module 4 closest to, the thioester domain, placing subsequent modules of FH near to any surface to which C3b is attached. The data do not rule out flexibility of the thioester domain relative to the remainder of the complex., (Copyright © 2011 The Protein Society.)

Characterization of segmental flexibility is needed to understand the biological mechanisms of the very large category of functionally diverse proteins, exemplified by the regulators of complement activation, that consist of numerous compact modules or domains linked by short, potentially flexible, sequences of amino acid residues. The use of NMR-derived residual dipolar couplings (RDCs), in magnetically aligned media, to evaluate interdomain motion is established but only for two-domain proteins. We focused on the three N-terminal domains (called CCPs or SCRs) of the important complement regulator, human factor H (i.e., FH1-3). These domains cooperate to facilitate cleavage of the key complement activation-specific protein fragment, C3b, forming iC3b that no longer participates in the complement cascade. We refined a three-dimensional solution structure of recombinant FH1-3 based on nuclear Overhauser effects and RDCs. We then employed a rudimentary series of RDC data sets, collected in media containing magnetically aligned bicelles (disklike particles formed from phospholipids) under three different conditions, to estimate interdomain motions. This circumvents a requirement of previous approaches for technically difficult collection of five independent RDC data sets. More than 80% of conformers of this predominantly extended three-domain molecule exhibit flexions of <40°. Such segmental flexibility (together with the local dynamics of the hypervariable loop within domain 3) could facilitate recognition of C3b via initial anchoring and eventual reorganization of modules to the conformation captured in the previously solved crystal structure of a C3b:FH1-4 complex.

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 CR1's 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 soluble 155 kDa glycoprotein factor H (FH) protects host tissue from damage by the human complement system. It accelerates decay of the alternative-pathway C3 convertase, C3bBb, and is a cofactor for factor I-mediated cleavage of the opsonin C3b. Numerous mutations and single-nucleotide polymorphisms (SNPs) occur in the gene encoding FH and the resulting missense mutations and truncation products result in altered functionality that predisposes to the development of the serious renal condition atypical haemolytic uraemic syndrome (aHUS). Other polymorphisms are linked to membranoproliferative glomerulonephritis and macular degeneration. The two C-terminal modules of FH (FH19-20) harbour numerous aHUS-associated mutations that disrupt the ability of factor H to protect host cells from complement-mediated damage. In this work, the crystal structure of an aHUS-associated T1184R variant of FH19-20 at a resolution of 1.52 Å is described. It is shown that this mutation has negligible structural effects but causes a significant change in the electrostatic surface of these two domains. Mechanisms are discussed by which this mutation may alter FH-ligand interactions, particularly with regard to the extension of a region of this molecule within module 20 that has been associated with the binding of glycosaminoglycans (GAGs) or sialic acid residues.

Human complement factor H (FH), an abundant 155-kDa plasma glycoprotein with 40 disulphide bonds, regulates the alternative-pathway complement cascade. Mutations and single nucleotide polymorphisms in the FH gene predispose to development of age-related macular degeneration, atypical haemolytic uraemic syndrome and dense deposit disease. Supplementation with FH variants protective against disease is an enticing therapeutic prospect. Current sources of therapeutic FH are restricted to human blood plasma highlighting a need for recombinant material. Previously FH expression in cultured plant, mammalian or insect cells yielded protein amounts inadequate for full characterisation, and orders of magnitude below therapeutic usefulness. Here, the V62,Y402 variant of FH has been produced recombinantly (rFH) in Pichia pastoris cells. Codon-optimisation proved essential whilst exploitation of the yeast mating α-factor peptide ensured secretion. We thereby produced multiple 10s-of-milligram of rFH. Following endoglycosidase H digestion of N-linked glycans, rFH (with eight residual N-acetylglucosamine moieties) was purified on heparin-affinity resin and anion-exchange chromatography. Full-length rFH was verified by mass spectrometry and Western blot using monoclonal antibodies to the C-terminus. Recombinant FH is a single non-aggregated species (by dynamic light scattering) and fully functional in biochemical and biological assays. An additional version of rFH was produced in which eight N-glycosylation sequons were ablated by Asn-Gln substitutions resulting in a glycan-devoid product. Successful production of rFH in this potentially very highly expressing system makes production of therapeutically useful quantities economically viable. Furthermore, ease of genetic manipulation in P. pastoris would allow production of engineered FH versions with enhanced pharmacokinetic and pharmacodynamic properties., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Many mutations associated with atypical hemolytic uremic syndrome (aHUS) lie within complement control protein modules 19-20 at the C terminus of the complement regulator factor H (FH). This region mediates preferential action of FH on self, as opposed to foreign, membranes and surfaces. Hence, speculation on disease mechanisms has focused on deficiencies in regulation of complement activation on glomerular capillary beds. Here, we investigate the consequences of aHUS-linked mutations (R53H and R78G) within the FH N-terminal complement control protein module that also carries the I62V variation linked to dense-deposit disease and age-related macular degeneration. This module contributes to a four-module C3b-binding site (FH1-4) needed for complement regulation and sufficient for fluid-phase regulatory activity. Recombinant FH1-4(V62) and FH1-4(I62) bind immobilized C3b with similar affinities (K(D) = 10-14 μM), whereas FH1-4(I62) is slightly more effective than FH1-4(V62) as cofactor for factor I-mediated cleavage of C3b. The mutant (R53H)FH1-4(V62) binds to C3b with comparable affinity (K(D) ∼12 μM) yet has decreased cofactor activities both in fluid phase and on surface-bound C3b, and exhibits only weak decay-accelerating activity for C3 convertase (C3bBb). The other mutant, (R78G)FH1-4(V62), binds poorly to immobilized C3b (K(D) >35 μM) and is severely functionally compromised, having decreased cofactor and decay-accelerating activities. Our data support causal links between these mutations and disease; they demonstrate that mutations affecting the N-terminal activities of FH, not just those in the C terminus, can predispose to aHUS. These observations reinforce the notion that deficiency in any one of several FH functional properties can contribute to the pathogenesis of this disease.

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.

Jagged-1, one of the five Notch ligands in man, is a membrane-spanning protein made of a large extracellular region and a 125-residue cytoplasmic tail bearing a C-terminal PDZ recognition motif ((1213) RMEYIV(1218) ). Binding of Jagged-1 intracellular region to the PDZ domain of afadin, a protein located at cell-cell adherens junctions, couples Notch signaling with the adhesion system and the cytoskeleton. Using NMR chemical shift perturbation and surface plasmon resonance, we studied the interaction between the PDZ domain of afadin (AF6&#95;PDZ) and a series of polypeptides comprising the PDZ-binding motif. Chemical shift mapping of AF6&#95;PDZ upon binding of ligands of different length (6, 24, and 133 residues) showed that the interaction is strictly local and involves only the binding groove in the PDZ. The recombinant protein corresponding to the entire intracellular region of Jagged-1, J1&#95;ic, is mainly disordered in solution, and chemical shift mapping of J1&#95;ic in the presence of AF6&#95;PDZ showed that binding is not coupled to folding. Binding studies on a series of 24-residue peptides phosphorylated at different positions showed that phosphorylation of the tyrosine at position -2 of the PDZ-binding motif decreases its affinity for AF6&#95;PDZ, and may play a role in the modulation of this interaction. Finally, we show that the R1213Q mutation located in the PDZ-binding motif and associated with extrahepatic biliary atresia increases the affinity for AF6&#95;PDZ, suggesting that this syndrome may arise from an imbalance in the coupling of Notch signaling to the cytoskeleton., (Copyright © 2010 John Wiley & Sons, Ltd.)