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Case Reports
. 2018 Jul;142(1):311-314.e6.
doi: 10.1016/j.jaci.2018.01.048. Epub 2018 Mar 6.

A type III complement factor D deficiency: Structural insights for inhibition of the alternative pathway

Christopher C T Sng  1 , Sorcha O'Byrne  2 , Daniil M Prigozhin  3 , Matthias R Bauer  4 , Jennifer C Harvey  5 , Michelle Ruhle  6 , Ben G Challis  7 , Sara Lear  2 , Lee D Roberts  8 , Sarita Workman  5 , Tobias Janowitz  1 , Lukasz Magiera  1 , Rainer Doffinger  2 , Matthew S Buckland  5 , Duncan J Jodrell  1 , Robert K Semple  9 , Timothy J Wilson  10 , Yorgo Modis  3 , James E D Thaventhiran  11
Affiliations
Case Reports

A type III complement factor D deficiency: Structural insights for inhibition of the alternative pathway

Christopher C T Sng et al. J Allergy Clin Immunol. 2018 Jul.
No abstract available

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Figures

Fig 1
Fig 1
Assessing the contribution of mutation R176P to AP dysfunction. A, Alternative pathway hemolytic activity (AP50) assay assessing patient serum supplemented with properdin (P), FB, or FD. B, The immediate family pedigree of the patient with the CFD genotype, serum AP50, and serum FD concentrations displayed. D represents the WT allele and d, the mutant allele (c.602G>C). C, Thermal shift assay of WT and R176P FD. D, Serial dilutions of recombinant WT or R176P FD were incubated with C3b and FB. The SDS-PAGE gel, stained with AcquaStain, shows the individual proteins and resultant products.
Fig 2
Fig 2
Defining the effects of the R176P mutation on FD function. A and B, FB-binding exosite loop 155-167 assumes a new conformation in mutant FD simulation. Arrows highlight average Cα position shifts of 2 residues that bind C3bB in the R176P FD simulation. C, Loss of shape complementarity at the FD-C3bB interface. FD exosite loops from published cocrystal structures (white, Protein Data Bank ID: 2XWB) overlaid with the simulated loops of WT and mutant FD. D, Distance sampled between the active site Nε2 nitrogen of His41 and Oγ of Ser183 during each simulation. The shorter distance is necessary for catalytic activity. E and F, Surface plasmon resonance binding measurement of enzymatically inactive recombinant FD (R176P/S183A or WT/S183A) to C3bB complex. G, Steady state kinetics for Z-Lys-SBzl cleavage by WT, R176P, R176A, and catalytically inactive control S183A FD. RMSD, Root-mean-square deviation.
Fig E1
Fig E1
Mutation R176P results in a type III FD deficiency. A, Chromatograms for the DNA sequence adjacent to position c.602 are shown for each member of the pedigree. The identified variant is rare: the Exome Aggregation Consortium's ExAC database reports mutation R176P (variant 19:861943 G/C) at an allele frequency of 1.049 × 10−4, with no homozygotes.B, Western blot analysis of FD in serum from the patient and healthy control. C, Secondary structural compositions of WT and R176P FD were evaluated using circular dichroism spectroscopy. D, Comparison of in vitro catalytic activity of recombinant WT, R176P, R176Q, and R176A FD in terms of FB cleavage. ***P < .001; ****P < .0001. E, Recombinant WT and R176P FD were tested for the ability to reconstitute AP50 when added to FD-depleted serum.
Fig E2
Fig E2
Mutation R176P stabilizes the self-inhibited state of FD. A, Structure of free FD (PDB ID: 2XW9) showing the catalytic triad (Ser183-His41-Asp89) in an inactive conformation stabilized by the self-inhibitory loop 199-202 (red) and an ion bridge between Asp177 and Agr202. The exosite loops are shown in yellow. B, Structure of C3bB-bound FD (PDB ID: 2XWB) omitting the C3b and FB components. FD exosite loops retain a conformation similar to that of unbound FD. C, WT, R176P, and R176A structures were stable over 100 nanoseconds of unrestrained molecular dynamics simulation with explicit solvent. D, Root mean square fluctuation (RMSF) in WT and mutant FD over the second half of the trajectory. E, Differences in WT versus R176 RMSF mapped to the FD structured. MD predicted increased mobility in exosite loops, notably 155-167, and decreased mobility in loops carrying the catalytic His41 and Asp89 residues.
Fig E3
Fig E3
Assessment of glucose tolerance in patients with functional FD deficiency. Patient and sibling were given 75 g of oral glucose at 0 minutes and blood glucose was measured at regular intervals between 0 and 120 minutes. The error bars indicate the range of plasma glucose concentrations between the patient and sibling.
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References

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