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Title: The Structure of the Cell-Wall Protease from Streptococci that Inactivates the Human Complement Factor 5A

Abstract

The structure of a 949-residue fragment of complement factor 5a peptidase (SCP) was determined to 1.9 Angstroms resolution. The molecule is made of five distinct domains in an elongated head-stalk structure. The structure suggests that activity of SCP can be modulated through binding of integrins to 2 RGD sequences. This structure is the first of an enzyme that is covalently attached to the cell wall of a Gram-positive bacteria. SCP is also the first functional protease containing a protease-associated domain to have its structure elucidated.

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
914264
Report Number(s):
BNL-78832-2007-JA
TRN: US200809%%125
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Int. Congr. Ser.; Journal Volume: 1289
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; BACTERIA; CELL WALL; ENZYMES; FUNCTIONALS; RESOLUTION; PROTEIN STRUCTURE; national synchrotron light source

Citation Formats

Brown,C., Gu, Z., Matsuka, Y., Olmsted, S., Cleary, P., Ohlendorf, D., and Earhart, C.. The Structure of the Cell-Wall Protease from Streptococci that Inactivates the Human Complement Factor 5A. United States: N. p., 2006. Web. doi:10.1016/j.ics.2005.11.004.
Brown,C., Gu, Z., Matsuka, Y., Olmsted, S., Cleary, P., Ohlendorf, D., & Earhart, C.. The Structure of the Cell-Wall Protease from Streptococci that Inactivates the Human Complement Factor 5A. United States. doi:10.1016/j.ics.2005.11.004.
Brown,C., Gu, Z., Matsuka, Y., Olmsted, S., Cleary, P., Ohlendorf, D., and Earhart, C.. Sun . "The Structure of the Cell-Wall Protease from Streptococci that Inactivates the Human Complement Factor 5A". United States. doi:10.1016/j.ics.2005.11.004.
@article{osti_914264,
title = {The Structure of the Cell-Wall Protease from Streptococci that Inactivates the Human Complement Factor 5A},
author = {Brown,C. and Gu, Z. and Matsuka, Y. and Olmsted, S. and Cleary, P. and Ohlendorf, D. and Earhart, C.},
abstractNote = {The structure of a 949-residue fragment of complement factor 5a peptidase (SCP) was determined to 1.9 Angstroms resolution. The molecule is made of five distinct domains in an elongated head-stalk structure. The structure suggests that activity of SCP can be modulated through binding of integrins to 2 RGD sequences. This structure is the first of an enzyme that is covalently attached to the cell wall of a Gram-positive bacteria. SCP is also the first functional protease containing a protease-associated domain to have its structure elucidated.},
doi = {10.1016/j.ics.2005.11.004},
journal = {Int. Congr. Ser.},
number = ,
volume = 1289,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • The human complement factor I gene (IF) was cloned from a flow-sorted cosmid library. The gene spans 63 kb and comprises 13 exons. The first exon, which encodes the leader sequence and 5{prime} untranslated region, is separated from the body of the gene by a large intron of 36 kb. Factor I is a mosaic protein. The second exon encodes a module found only in complement C6 and C7 (FI/C6/C7); the third and fourth exons each encode a low-density lipoprotein receptor module. Two very small exons, 21 and 36 bp, then separate the first six exons from the last fivemore » that encode the serine protease domain of factor I. Within the serine protease gene family factor I has a unique genomic structure, but it bears a much closer resemblance to trypsin than it does to the other complement system serine proteases, factor B, C2, and C1r/C1s. 58 refs., 3 figs., 2 tabs.« less
  • The human pre-monocytic cell line U-937 was shown to synthesize and to secrete increasing amounts of factor B in short term cultures in serum-free medium containing BSA. The kinetics of factor B production were higher on day 2 than on days 1 and 3. The production of factor B was reversibly inhibited by cycloheximide, indicating de novo synthesis. Metabolic labeling with (/sup 35/S)-methionine and SDS-PAGE analysis revealed that both intracellular and secreted factor B were single-chain proteins with similar m.w. (90,000), which co-migrated with purified plasma factor B. Incubation of U-937 cells with the immunostimulants PMA, LPS, IFN-gamma, and IL-1more » resulted in a dose-dependent augmentation of factor B production. A 24-h exposure to IL-1 was shown to be required for maximal stimulation. A combination of suboptimal doses of LPS and IFN-gamma was shown to exert a synergistic effect on factor B production. The U-937 cell line is thus a valuable model for the study of the regulation of the factor B gene expression.« less
  • Factor H is a regulatory component of the complement system. It has a monomer M{sub r} of 150,000. Primary structure analysis shows that the polypeptide is divided into 20 homologous regions, each 60 amino acid residues long. These are independently folding domains and are termed short consensus repeats (SCRs) or complement control protein (CCP) repeats. High-flux synchrotron x-ray and neutron scatteriing studies were performed in order to define its solution structure in conditions close to physiological. The M{sub r} of factor H was determined as 250,000-320,000 to show that factor H is dimeric. The radius of gyration R{sub G} ofmore » native factor H by X-rays or by neutrons in 0% or 100% {sup 2}H{sub 2}O buffers is not measurable but is greater than 12.5 nm. Two cross-sectional radii of gyration R{sub XS-1} and R{sub XS-2} were determined as 3.0-3.1 and 1.8 nm, respectively. Analyses of the cross-sectional intensities show that factor H is composed of two distinct subunits. This model corresponds to an actual R{sub G} fo 21-23 nm. The separation between each SCR/CCP in factor H is close to 4 nm. In the solution structure of factor H, the SCR/CCP domains are in a highly extended conformation.« less
  • Binding of /sup 125/I-labeled ..cap alpha../sub 2/-macroglobulin (..cap alpha../sub 2/M) to streptococci belonging to serological groups A, B, C, and G was studied. Streptococci of groups A and G interacted only with native ..cap alpha../sub 2/M, and those of group C reacted only with ..cap alpha../sub 2/M-trypsin complex. Binding of ..cap alpha../sub 2/M to group A streptococci was saturable and reversible. The dissociation constant was 2.02 x 10/sup -7/ M, and the number of binding sites was calculated to be 18,000 per streptococcus. The ..cap alpha../sub 2/M-binding protein could be solubilized by treatment of group A streptococci with a murolyticmore » enzyme and subsequently purified by affinity chromatography and high-pressure liquid chromatography. The purified protein was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 78,000. It possessed no proteolytic activity and interacted with native ..cap alpha../sub 2/M in Western blots (immunoblots). Interaction of purified binding protein with ..cap alpha../sub 2/M led to a change in the conformation of ..cap alpha../sub 2/M similar to that obtained by ..cap alpha../sub 2/M-protease complexes. Reversible binding of a nonproteolytic streptococcal component of ..cap alpha../sub 2/M is thus a novel feature of ..cap alpha../sub 2/M reactivity.« less