Modelling of the serine-proteinase fold by X-ray and neutron scattering and sedimentation analyses: occurrence of the fold in factor D of the complement system.

ABSTRACT: Solution scattering is a powerful means of determining the overall arrangement of domains in the multidomain proteins of complement. the serine-proteinase domain is central to all proteolytic events during complement activation. As models of this domain, bovine beta-trypsin, trypsinogen, alpha-chymotrypsin and chymotrypsinogen A were studied by neutron and X-ray synchrotron solution scattering. At pH 7, all the X-ray and neutron M(r) values corresponded to monomeric proteins. The X-ray radii of gyration, RG, of beta-trypsin, trypsinogen, alpha-chymotrypsin and chymotrypsinogen A (measured in positive solute-solvent contrasts) were 1.59 nm, 1.78 nm, 1.71 nm and 1.76 nm (+/- 0.05-0.11 nm) in that order. Neutron contrast variation showed that the RG at infinite contrast, RC, for these four proteins were 1.57 nm, 1.70 nm, 1.67 nm and 1.78 nm (+/- 0.03 nm) in that same order. The radial inhomogeneity of neutron-scattering density, alpha, was positive at (5-13) x 10(-5), and corresponds to the preponderance of hydrophilic residues near the protein surface. On trypsinogen activation, a small reduction in the RG value of 0.13 +/- 0.07 nm was just detectable, while the RG of chymotrypsinogen A was unchanged after activation. The RC and alpha values of the four proteins can be calculated by using crystallographic co-ordinates. The reduced RG of beta-trypsin relative to trypsinogen was explained in terms of the removal of the extended N-terminal hexapeptide of trypsinogen. The full X-ray and neutron-scattering curves in positive and negative contrasts agreed well with scattering curves calculated from crystallographic coordinates to a nominal structural resolution of 4.5 nm, provided that the internal structure was considered in neutron modelling, and that the hydration was considered in X-ray modelling. Sedimentation-coefficient data also provide information on the disposition of domains in multidomain proteins. It was found that the hydrated X-ray sphere model could be directly utilized to calculate sedimentation coefficients. X-ray scattering on factor D showed from its RG of 1.78 nm that this is monomeric and very similar in structure to beta-trypsin. The X-ray-scattering curve of factor D was readily modelled using the beta-trypsin crystal structure after allowance for sequence changes. The success of these modellings provides a basis for the constrained modelling of solution scattering data for the multidomain proteins of complement.

SUBMITTER: Perkins SJ 

PROVIDER: S-EPMC1134824 | biostudies-other | 1993 Oct

REPOSITORIES: biostudies-other