The role of strand 1 of the C beta-sheet in the structure and function of alpha(1)-antitrypsin.
Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C beta-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant alpha(1)-antitrypsin Pittsburgh mutant (P1 = Arg). Mutation of the P10' position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7' and P9' caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6' and P8' positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6' and P8' residues of the C beta-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6' and P8' mutants of alpha(1)-antitrypsin had melting temperatures 14 degrees lower than wild-type alpha(1)-antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants.
Bottomley
S P
SP
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Lawrenson
I D
ID
Tew
D
D
Dai
W
W
Whisstock
J C
JC
Pike
R N
RN
D000991:Antithrombins / Q000737:chemistry; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D015843:Serpins / Q000737:chemistry; D013696:Temperature; D013917:Thrombin / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000502:physiology*
D000991:Antithrombins; D015843:Serpins; D000515:alpha 1-Antitrypsin; D013917:Thrombin