Interaction between the P14 residue and strand 2 of beta-sheet B is critical for reactive center loop insertion in plasminogen activator inhibitor-2.
The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.
Saunders
D N
DN
Department of Biological Sciences, University of Wollongong, Northfields Ave, Wollongong 2522, Australia. d.saunders@garvan.org.au
Jankova
L
L
Harrop
S J
SJ
Curmi
P M
PM
Gould
A R
AR
Ranson
M
M
Baker
M S
MS
D000596:Amino Acids / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006860:Hydrogen Bonding; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D017396:Plasminogen Activator Inhibitor 2 / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013912:Threonine / Q000737:chemistry; D014508:Urea / Q000494:pharmacology
D000596:Amino Acids; D010455:Peptides; D017396:Plasminogen Activator Inhibitor 2; D011994:Recombinant Proteins; D013912:Threonine; D014508:Urea