Central modules of the vaccinia virus complement control protein are not in extensive contact.
The 28.6 kDa vaccinia virus complement control protein (VCP) is an inhibitor of the complement system and has therapeutic potential. It is composed of four domains or modules and is a homologue of complement receptor 1 (CR1) and other mammalian regulators of complement activation. A key aspect to structure-function relationships in these proteins is the extent of intramolecular module-module interactions, since these dictate the overall shape and flexibility of the molecules. A protein fragment (VCP approximately 2,3) encompassing modules 2 and 3 of VCP was over-expressed in Pichia pastoris. Ultracentrifugation showed that VCP approximately 2,3 is highly asymmetric with an axial ratio of 5.3:1, which is consistent with an end-to-end arrangement of the two modules. NMR spectroscopy, differential scanning calorimetry, CD and intrinsic tryptophan fluorescence were used to monitor unfolding of VCP approximately 2,3. Experiments performed over a range of temperatures and concentrations of guanidinium chloride revealed that module 2 unfolds under milder conditions than, and independently of, module 3. Unfolding of module 2 is not associated with extensive changes in amide (15)N and (1)H chemical shifts of module 3, implying that the modules do not form an extensive intermodular interface. Results obtained in this work for VCP approximately 2,3 are compared with those obtained in a study of CR1 modules 15-17 [Kirkitadze, Krych, Uhrin, Dryden, Smith, Cooper, Wang, Hauhart, Atkinson and Barlow (1999) Biochemistry 38, 7019-7031].
Kirkitadze
M D
MD
Edinburgh Centre for Protein Technology, Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, Scotland, U.K. marina@chem.ed.ac.uk
Henderson
C
C
Price
N C
NC
Kelly
S M
SM
Mullin
N P
NP
Parkinson
J
J
Dryden
D T
DT
Barlow
P N
PN
D000595:Amino Acid Sequence; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003169:Complement Inactivator Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D017124:Conserved Sequence; D019791:Guanidine; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011951:Receptors, Complement / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation; D014616:Vaccinia virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
D003169:Complement Inactivator Proteins; D011951:Receptors, Complement; D014764:Viral Proteins; C066459:complement-control protein, Vaccinia virus; D019791:Guanidine