Abstract
Factor I-like modules (FIMs) of complement proteins C6, C7, and factor I participate in protein-protein interactions critical to the progress of a complement-mediated immune response to infections and other trauma. For instance, the carboxyl-terminal FIM pair of C7 (C7-FIMs) binds to the C345C domain of C5 and its activated product, C5b, during self-assembly of the cytolytic membrane-attack complex. FIMs share sequence similarity with follistatin domains (FDs) of known three-dimensional structure, suggesting that FIM structures could be reliably modeled. However, conflicting disulfide maps, inconsistent orientations of subdomains within FDs, and the presence of binding partners in all FD structures led us to determine the three-dimensional structure of C7-FIMs by NMR spectroscopy. The solution structure reveals that each FIM within C7 contains a small amino-terminal FOLN subdomain connected to a larger carboxyl-terminal KAZAL domain. The open arrangement of the subdomains within FIMs resembles that of first FDs within structures of tandem FDs but differs from the more compact subdomain arrangement of second or third FDs. Unexpectedly, the two C7-FIMs pack closely together with an approximate 2-fold rotational symmetry that is rarely seen in module pairs and has not been observed in FD-containing proteins. Interfaces between subdomains and between modules include numerous hydrophobic and electrostatic contributions, suggesting that this is a physiologically relevant conformation that persists in the context of the parent protein. Similar interfaces were predicted in a homology-based model of the C6-FIM pair. The C7-FIM structures also facilitated construction of a model of the single FIM of factor I.
Highlights
Factor I-like modules (FIMs) of complement proteins C6, C7, and factor I participate in protein-protein interactions critical to the progress of a complement-mediated immune response to infections and other trauma
The initial residues of FIM2 (Cys773, Gly774, Pro777, and Leu778), which lie adjacent to the intermodular linker, form a solvent-exposed loop that did not converge during structure calculations because of the low number of NOEs observed in this region; this is reflected in the high root mean square deviations (RMSDs) of the relevant C␣ coordinates (Fig. 2b)
A useful first step toward a full molecular description of the early steps in membrane attack complex (MAC) formation is to elucidate the three-dimensional structures of FIM pairs at the carboxyl termini of C6 and C7
Summary
C7-FIMs, encompassing residues Asn693 to Gln843 of C7 with an amino-terminal His tag, was cloned previously (18), and the recombinant protein was expressed in the Origami B strain of Escherichia coli as previously described (18, 28). The incidence of hydrogen bonds between backbone amides and carboxyl groups was determined on the basis of amide exchange retardation as observed in 15N,1H HSQC spectra collected 1 h after the protein was transferred to 99.9% (v/v) deuterated buffer. B, stereo image of the backbone overlay of the ensemble of 25 lowest NOE energy, NMR-derived structures of C7-FIMs of 100 structures calculated. The three-dimensional structure of C6-FIMs was modeled using, as a template, the structure of C7-FIMs closest to that of the mean from the ensemble generated in structure calculations. The resulting target-template alignments were manually refined from the TABLE 1 multiple sequence alignment to place gaps optimally guided by Structural statistics for the lowest energy structures positioning of predicted and identified secondary structure ele-. FIM1 is defined as 701–767, and FIM2 is defined as 772– 841, excluding residues adjacent to the linker: 773, 774, 777, and 778, and in the apical -hairpin: 783–787
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