Although protein serine proteinase inhibitors of the Kunitz, Kazal, and Bowman-Birk families make do with lock-and-key type interactions to inhibit target proteinases, serpins have now been shown by x-ray crystallography, NMR spectroscopy and FRET to employ a remarkable conformational change-based mechanism to inhibit some of the same serine proteinases. This mechanism involves changes to both the serpin and the proteinase, with insertion of the serpin reactive center loop (RCL) into the center of its own b-sheet A, and translocation of the proteinase over 70A to the distal end of the serpin from its initial docking position, with concomitant distortion of the proteinase active site and a """"""""crushing"""""""" of a large portion of the proteinase upon it reaching its final location. Whereas these studies have provided a structural explanation of how serpins inhibit serine proteinases, they have not explained why such a complex, error-prone, mechanism is employed. An unproven assumption is that the conformational changes that occur in both serpin and proteinase are exploited subsequently for other downstream events, whether recognition and signaling via a receptor such as LRP, or else rendering the proteinase incapable of revival, through cleavage while in the serpin-proteinase complex. We propose three specific aims that together will enable evaluation of the importance of the full proteinase-translocating, proteinase-crushing sequence in expression of the functions that make serpins the preferred class of proteinase inhibitors under many circumstances.
Specific Aim 1 will determine the structural requirements and functional consequences of serpin and proteinase binding to the receptor LRP.
Specific Aim 2 will determine the structure of the mosquito serpin AFXa, its mechanism of inhibition of human factor Xa and the properties of the AFXa-Xa complex in relation to LRP binding and signaling.
Specific Aim 3 will determine the conformational changes that occur in caspases, cathepsins and subtilisin-like proteinases upon formation of covalent complexes with serpins. We will use thermodynamic measurements of binding affinity and structural approaches of NMR spectroscopy, FRET and x-ray crystallography that we have successfully used in the previous grant period and, where appropriate, correlate our findings with functional consequences, through a collaboration with Dr. Dudley Strickland.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL079430-04
Application #
7331510
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
2004-12-27
Project End
2009-11-30
Budget Start
2007-12-01
Budget End
2008-11-30
Support Year
4
Fiscal Year
2008
Total Cost
$367,420
Indirect Cost
Name
University of Illinois at Chicago
Department
Biochemistry
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Gettins, Peter G W; Olson, Steven T (2016) Inhibitory serpins. New insights into their folding, polymerization, regulation and clearance. Biochem J 473:2273-93
Huang, Xin; Dementiev, Alexey; Olson, Steven T et al. (2010) Basis for the specificity and activation of the serpin protein Z-dependent proteinase inhibitor (ZPI) as an inhibitor of membrane-associated factor Xa. J Biol Chem 285:20399-409
Jensen, Jan K; Dolmer, Klavs; Schar, Christine et al. (2009) Receptor-associated protein (RAP) has two high-affinity binding sites for the low-density lipoprotein receptor-related protein (LRP): consequences for the chaperone functions of RAP. Biochem J 421:273-82
Jensen, Jan K; Dolmer, Klavs; Gettins, Peter G W (2009) Specificity of binding of the low density lipoprotein receptor-related protein to different conformational states of the clade E serpins plasminogen activator inhibitor-1 and proteinase nexin-1. J Biol Chem 284:17989-97
Gettins, Peter G W; Olson, Steven T (2009) Exosite determinants of serpin specificity. J Biol Chem 284:20441-5
Jensen, Jan K; Gettins, Peter G W (2008) High-resolution structure of the stable plasminogen activator inhibitor type-1 variant 14-1B in its proteinase-cleaved form: a new tool for detailed interaction studies and modeling. Protein Sci 17:1844-9
Swanson, Richard; Raghavendra, Manikanahally P; Zhang, Weiqing et al. (2007) Serine and cysteine proteases are translocated to similar extents upon formation of covalent complexes with serpins. Fluorescence perturbation and fluorescence resonance energy transfer mapping of the protease binding site in CrmA complexes with granzyme J Biol Chem 282:2305-13