Kinetic Stability of Bacterial Proteases

A common assumption in protein folding is that native states of proteins are at a global energy minimum. Recently, we have shown that alpha-lytic protease (aLP) shatters this assumption. aLP is an extracellular bacterial serine protease of the chymotrypsin family that is synthesized as a pro-enzyme.

Both in vivo and in vitro, we have shown that the pro region (Pro) is absolutely required for correct folding of the protease domain either in cis (pro-enzyme) or in trans (Pro supplied as a separate polypeptide chain). Refolding chemically-denatured aLP in the absense of the pro region results in the formation of a stable molten globule folding intermediate (Int). Addition of pro leads to rapid formation of the native state suggeseting that, without Pro, folding of Int is blocked by a large kinetic barrier. Recently, we have been able to measure the rates of Int folding (t1/2 > 1000 years) and native state unfolding (t1/2 ~ 1 year). Remarkably, this demonstrates that the aLP native state is less stable than both the intermediate and the fully unfolded molecule (Fig 2). aLP stability arises soley from the kinetic barrier that blocks its unfolding and not from thermodynamics. As such, its folding is entirely dependent on the pro region which acts to both catalyze the folding reaction (rate acceleration = 30109) and to stabilize the native state during folding (Pro is a potent inhibitor of the enzyme, Ki ~ 10-10 M). Free aLP is released through proteolytic degredation of Pro.

Deletions of hte C-terminal residues of Pro demonstrates that these residues participate in preferentially stabilizing the rate limiting folding transition state. Using one such debilitated Pro (Pro-3) we carried out a genetic screen to look for aLP mutations that would increase the rate of folding. The result was a pair of mutations (R102H/G134S) that lowered the folding barrier by ~3.5 kcal/mol for both the Pro catalyzed and uncatalyzed reactions.

The crystal structure of the inhibitory complex between aLP and Pro (the product of the folding reaction) reveals that the C-shaped pro region completely surrounds the C-terminal domain of the aLP, making a 5-stranded b-sheet and placing the Pro C-terminus in the active site. Together with the suppressor mutations, this provides strong evidence that the folding defect is located in the C-domain. Current and future efforts focus on understanding the origin of the folding barrier and the mechanism of pro-mediated catalysis.

Relevant Publications

Brian A. Kelch, Kyle P. Eagen, F. Pinar Erciyas, Elisabeth L. Humphris, Adam R. Thomason, Shinji Mitsuiki and David A. Agard, "Structural and Mechanistic Exploration of Acid Resistance: Kinetic Stability Facilitates Evolution of Extremophilic Behavior," Journal of Molecular Biology, 2007 Mar (pdf).

Fuhrmann, C.N., Daugherty, M.D., and Agard, D.A., "Subangstrom Crystallography Reveals that Short Ionic Hydrogen Bonds, and Not a His-Asp Low-Barrier Hydrogen Bond, Stabilize the Transition State in Serine Protease Catalysis," JACS, 2006, 128(28), 9086-102. (pdf).

Truhlar, S.M.E. and Agard, D.A., "The Folding Landscape of an α-Lytic Protease Variant Reveals the Role of a Conserved Beta-Hairpin in the Development of Kinetic Stability." Proteins: Structure, Function, and Bioinformatics (2005), 61, 105-114. (pdf).

Jaswal SS, Truhlar SM, Dill KA, Agard DA., "Comprehensive analysis of protein folding activation thermodynamics reveals a universal behavior violated by kinetically stable proteases." J Mol Biol. 2005 Mar 25;347(2):355-66. (html or pdf).

Fuhrmann, C.N., Kelch, B.A., Ota, N., Agard, D.A. "The 0.83Å resolution crystal structure of α-lytic protease reveals the detailed structure of the active site and identifies a source of conformational strain," J. Mol Biol. (2004);338(5):999-1013. (html or pdf).

Stephanie M.E. Truhlar, Erin L. Cunningham and David A. Agard. The folding landscape of Streptomyces griseus protease B reveals the energetic costs and benefits associated with evolving kinetic stability. Protein Science (2004), 13:381-390 (pdf).

Erin L. Cunningham and David A. Agard. Disabling the folding catalyst is the last critical step in α-lytic protease folding. Protein Science (2004), 13:325-331(pdf).

Erin L. Cunningham and David A. Agard. Interdependent Folding of the N- and C-Terminal Domains Defines the Cooperative Folding of α-Lytic Protease. Biochemistry; 2003, 42, 13212-13219 (html and pdf).

Cunningham, E., Mau, T., Truhlar, S.M.E., Agard, D.A. (2002). The Pro Region N-Terminal Domain Provides Specific Interactions Required for Catalysis of α-Lytic Protease Folding. Biochemistry 41(28), 8860-7, (html or pdf).

Jaswal, Sheila S.; Sohl, Julie L. Davis, Jonathan H.; Agard, David A. "Energetic landscape of α-lytic protease optimzes longevity through kinetic stability." Nature 415 (2002) 343-6 (pdf).

Ota, N., Agard, D.A. (2001) Enzyme specificity under dynamic control II: Principal component analysis of α-lytic protease using global and local solvent boundary conditions. Protein Sci. 2001 Jul;10(7):1403-14 (pdf).

Derman, A.I., Agard, D.A. (2000) Two energetically disparate folding pathways of α-lytic protease share a single transition state. Nat Struct Biol. 7(5):394-7 (pdf).

Cunningham, E.L., Jaswal, S.S., Sohl J.L., Agard D.A. (1999) Kinetic stability as a mechanism for protease longevity. Proc Natl Acad Sci U S A. 96(20):11008-14 (pdf).

Miller, D.W. and Agard, D.A. (1999). Enzyme Specificity Under Dynamic Control: A Normal Mode Analysis of α-Lytic Protease. Journal of Molecular Biology 286(1); 267-78 (pdf).

Anderson, D.E., Peters, R.J., Wilk, B., Agard, D.A. (1999). α-lytic protease precursor: characterization of a structured folding intermediate. Biochemistry 38(15); 4728-35 (pdf).

Sauter, N.K., Mau, T., Rader, S.D., and Agard, D.A. (1998). Molecular architecture of a folding catalyst:α-lytic protease complexed with its pro region. Nature Structural Biology 5: 945-950 (pdf).

Sohl, J.L., Jaswal, S.S., and Agard, D.A. (1998). Unfolded conformations are more stable than the native state of α-lytic protease. Nature 395: 817-819 (pdf).

Peters, R.J., Shiau, A.K., Sohl, J.L., Anderson, D.E., Tang, G., Silen, J.L., Agard, D.A. (1998). Pro region C-terminus: protease active site interactions are critical in catalyzing the folding of α-lytic protease. Biochemistry. 37:12058-12067 (pdf).

Davis, J.H. and Agard, D.A. (1998). Relationship between enzyme specificity and the backbone dynamics of free and inhibited α-lytic protease. Biochemistry. 37:7696-7707 (pdf).

Davis, Jonathan H.; Agard, David A.; Handel, Tracy M.; Basus, Vladimir J. Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to α-lytic protease. Journal of Biomolecular NMR (1997), 10(1), 21-27. (pdf).

Rader, S.D. and Agard, D.A. (1997). Conformational substrates in enzyme mechanism: the 120K structure of α-lytic protease at 1.5 Å resolution. Protein Science 6: 1375-1386. (pdf).

Sohl, J.L.α-lytic protease by pro region c-terminal steric occlusion of the active site. Biochemistry 36(13): 3894-3902 (pdf).

Boggs, Amy Fujishige; Agard, David A.. Bacterial extracellular secretion: transport of α-lytic protease across the outer membrane of Escherichia coli. Membrane Protein Transport (1996), 3 165-179. (pdf).

Mace, J.E. and Agard, D.A. (1995). Kinetic and structural characterization of mutations of glycine 216 in α-lytic protease: a new target for engineering substrate specificity. J. Mol. Biol. 254: 720-736.(pdf).

Mace, J.E., Wilk, B.J., and Agard, D.A. (1995). Functional linkage between the active site of α-lytic protease and distant regions of structure: scanning alanine mutagenesis of a surface loop affects activity and substrate specificity. J. Mol. Biol. 251: 116-134.(pdf).

Baker, D., Shiau, A.K., and Agard, D.A. (1993). The role of pro regions in protein folding. The role of pro regions in protein folding. Curr. Opinion Cell Biol. 5: 966-970. (pdf).

Fujishige, A., Smith, K., Silen, J.L., and Agard, D.A. (1992). Correct folding of α-lytic protease is required for its extracellular secretion from E. coli. J.C.B. 118:33-42. (pdf).

Baker, D., Sohl, J.L., and Agard, D.A. (1992). A protein-folding reaction under kinetic control. Nature 356: 263-265. (pdf).

Baker, D., Silen, J.L., and Agard, D.A. (1992). Protease pro region required for folding is a potent inhibitor of the mature enzyme. Proteins 12: 339-344.

Bone R; Sampson N S; Bartlett P A; Agard D A, Crystal structures of α-lytic protease complexes with irreversibly bound phosphonate esters. Biochemistry (1991), 30(8), 2263-72.(pdf).

Bone, R., Fujishige, A., Kettner, C.A., and Agard, D.A. (1991). Structural basis for broad specificity in α-lytic protease mutants. Biochem. 30: 10388-10398.(pdf).

Caldwell, J.W., Agard, D.A., and Kollman, P.A. (1991). Free energy calculations on binding and catalysis by α-lytic protease: the role of substrate size in the P1 pocket. Proteins 10: 140-148.

Silen, J.L. and Agard, D.A. (1989). The α-lytic protease pro region does not require a physical linkage to activate the protease domain in vivo. Nature 341: 462-464. (pdf).

Silen, J.L., Frank, D., Fujishige, A., Bone, R., and Agard, D.A. (1989). Analysis of prepro α-lytic protease expression in E. coli reveals that the pro region is required for activity. J. Bact. 171: 1320-1325.(pdf).

Silen, J.L., McGrath, C.N., Smith, K.R., and Agard, D.A. (1988). Molecular analysis of the gene encoding α-lytic protease: evidence for a preproenzyme. Gene 69: 237-244. (pdf).

Kettner, C.A., Bone, R., Agard, D.A., and Bachovchin, W.W. (1988). Kinetic properties of the binding of α-lytic protease to peptide boronic acids. Biochem. 27: 7682-7688.(pdf).

Bone, R., Shenvi, A.R., Kettner, C.A., and Agard, D.A. (1987). Serine protease mechanism: the structure of an inhibitory complex of α-lytic protease and a tightly bound peptide boronic acid. Biochem. 26: 7609-7614.(pdf).

Agard, D.A. and Stroud, R.M. (1982). α-bungarotoxin structure revealed by a rapid method or averaging electron density of non-crystallographically, translationally-related molecules. Acta Cryst. A38: 186-194.(pdf).