An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis
2016; Elsevier BV; Volume: 291; Issue: 51 Linguagem: Inglês
10.1074/jbc.m116.758417
ISSN1083-351X
AutoresOlumide Kayode, Ruiying Wang, Devon F. Pendlebury, Itay Cohen, Rachel D. Henin, Alexandra Hockla, Alexei S. Soares, Niv Papo, Thomas R. Caulfield, Evette S. Radisky,
Tópico(s)Enzyme Structure and Function
ResumoThe molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. Protein function is determined by macromolecular geometry conferred by the folded state, as noted by Anfinsen nearly 40 years ago (1.Anfinsen C.B. Principles that govern the folding of protein chains.Science. 1973; 181: 223-230Crossref PubMed Scopus (5135) Google Scholar); however, recent years have brought fresh meaning to this paradigm with an increasing appreciation of the temporal dependence of protein structure. Proteins constantly sample varied conformational fluctuations about the time-averaged structures that we observe crystallographically or spectroscopically, and these conformational dynamics are in many cases closely coupled to protein function (2.Frauenfelder H. Sligar S.G. Wolynes P.G. The energy landscapes and motions of proteins.Science. 1991; 254: 1598-1603Crossref PubMed Scopus (2609) Google Scholar, 3.Leeson D.T. Wiersma D.A. Looking into the energy landscape of myoglobin.Nat. Struct. Biol. 1995; 2: 848-851Crossref PubMed Scopus (61) Google Scholar4.Henzler-Wildman K. Kern D. Dynamic personalities of proteins.Nature. 2007; 450: 964-972Crossref PubMed Scopus (1710) Google Scholar). Nowhere is this clearer than for enzymes, proteins evolved to accelerate biological chemical reactions. Varied examples have revealed that enzyme conformational dynamics can facilitate substrate binding, progression along the catalytic reaction coordinate, and product release (5.Wolf-Watz M. Thai V. Henzler-Wildman K. Hadjipavlou G. Eisenmesser E.Z. Kern D. Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair.Nat. Struct. Mol. Biol. 2004; 11: 945-949Crossref PubMed Scopus (400) Google Scholar6.Boehr D.D. McElheny D. Dyson H.J. Wright P.E. The dynamic energy landscape of dihydrofolate reductase catalysis.Science. 2006; 313: 1638-1642Crossref PubMed Scopus (759) Google Scholar, 7.Watt E.D. Shimada H. Kovrigin E.L. Loria J.P. The mechanism of rate-limiting motions in enzyme function.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 11981-11986Crossref PubMed Scopus (137) Google Scholar, 8.Torbeev V.Y. Raghuraman H. Hamelberg D. Tonelli M. Westler W.M. Perozo E. Kent S.B. Protein conformational dynamics in the mechanism of HIV-1 protease catalysis.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 20982-20987Crossref PubMed Scopus (71) Google Scholar, 9.Bhabha G. Lee J. Ekiert D.C. Gam J. Wilson I.A. Dyson H.J. Benkovic S.J. Wright P.E. A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis.Science. 2011; 332: 234-238Crossref PubMed Scopus (363) Google Scholar10.Whittier S.K. Hengge A.C. Loria J.P. Conformational motions regulate phosphoryl transfer in related protein tyrosine phosphatases.Science. 2013; 341: 899-903Crossref PubMed Scopus (120) Google Scholar). Most studies of protein dynamics in enzyme catalysis have naturally focused on conformational changes within the enzyme. However, for the many enzymes that catalyze reactions of protein substrates, an overlooked source of potentially relevant dynamics lies within the substrate. Trypsins are serine proteases, a class of proteolytic enzymes that have been well characterized and used to dissect and understand catalysis, most often using short oligopeptide model substrates as proxies for natural protein substrates. Following formation of a noncovalent Michaelis complex, the catalytic mechanism proceeds through two sequential steps (Scheme 1) (11.Hedstrom L. Serine protease mechanism and specificity.Chem. Rev. 2002; 102: 4501-4524Crossref PubMed Scopus (1334) Google Scholar). In the first step, the enzyme serine nucleophile attacks the carbonyl of a substrate peptide bond, forming a covalent acyl-enzyme intermediate, and displacing the peptide product composed of residues C-terminal to the cleavage site (the "primed side" residues according to the nomenclature of Schechter and Berger (12.Schechter I. Berger A. On the size of the active site in proteases. I. Papain.Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4753) Google Scholar)). In the second step, a nucleophilic water molecule hydrolyzes the acyl-enzyme, releasing the nonprimed side peptide product. Classic studies using unstructured oligopeptide substrates have shown substrate discrimination to occur at the acylation step; for specific peptide substrates, acylation is fast, and acyl-enzyme hydrolysis is rate-determining (13.Hedstrom L. Szilagyi L. Rutter W.J. Converting trypsin to chymotrypsin: the role of surface loops.Science. 1992; 255: 1249-1253Crossref PubMed Scopus (451) Google Scholar, 14.Radisky E.S. Lee J.M. Lu C.J. Koshland Jr., D.E. Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 6835-6840Crossref PubMed Scopus (107) Google Scholar). Much less is known about cleavage kinetics for structured protein substrates, but overall rates of catalysis are often several orders of magnitude slower than for oligopeptide substrates (15.Salameh M.A. Robinson J.L. Navaneetham D. Sinha D. Madden B.J. Walsh P.N. Radisky E.S. The amyloid precursor protein/protease nexin 2 Kunitz inhibitor domain is a highly specific substrate of mesotrypsin.J. Biol. Chem. 2010; 285: 1939-1949Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), suggesting that substrate structure may variably impede progression of the reaction pathway. Furthermore, modeling studies have implicated a requirement for local unfolding of a protein chain around the cleavage site to enable proteolysis (16.Hubbard S.J. Eisenmenger F. Thornton J.M. Modeling studies of the change in conformation required for cleavage of limited proteolytic sites.Protein Sci. 1994; 3: 757-768Crossref PubMed Scopus (210) Google Scholar). Thus, it seems plausible that substrate conformational dynamics (i.e. the dynamical behavior of a protein substrate during catalysis) may influence serine protease catalytic rates. An ideal model to examine protein substrate dynamics and their impact on proteolysis is provided by the canonical (Laskowski mechanism) serine protease inhibitors (17.Krowarsch D. Cierpicki T. Jelen F. Otlewski J. Canonical protein inhibitors of serine proteases.Cell. Mol. Life Sci. 2003; 60: 2427-2444Crossref PubMed Scopus (182) Google Scholar). These inhibitors are highly specific limited proteolysis substrates for their target enzymes, yet they differ from ordinary substrates in being bound many orders of magnitude more tightly and cleaved many orders of magnitude more slowly (18.Laskowski Jr., M. Kato I. Protein inhibitors of proteinases.Annu. Rev. Biochem. 1980; 49: 593-626Crossref PubMed Scopus (1940) Google Scholar). They interact with an enzyme by mimicking an ideal substrate, using an exposed binding loop, which is complementary to the active site for recognition, and presenting a "reactive site" peptide bond for cleavage (19.Bode W. Huber R. Natural protein proteinase inhibitors and their interaction with proteinases.Eur. J. Biochem. 1992; 204: 433-451Crossref PubMed Scopus (1006) Google Scholar, 20.Radisky E.S. Koshland Jr., D.E. A clogged gutter mechanism for protease inhibitors.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10316-10321Crossref PubMed Scopus (130) Google Scholar). Intriguingly, short peptides that recapitulate the sequence surrounding the reactive site behave as ideal substrates, not inhibitors, and thus the slow cleavage and inhibitory nature of canonical inhibitors are conferred by structural context and perhaps consequent constraints on dynamics (21.Radisky E.S. King D.S. Kwan G. Koshland Jr., D.E. The role of the protein core in the inhibitory power of the classic serine protease inhibitor, chymotrypsin inhibitor 2.Biochemistry. 2003; 42: 6484-6492Crossref PubMed Scopus (27) Google Scholar). In studies using mesotrypsin, a trypsin isoform capable of more rapid proteolysis of canonical inhibitors compared with other trypsin family members (22.Szmola R. Kukor Z. Sahin-Tóth M. Human mesotrypsin is a unique digestive protease specialized for the degradation of trypsin inhibitors.J. Biol. Chem. 2003; 278: 48580-48589Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 23.Salameh M.A. Soares A.S. Hockla A. Radisky E.S. Structural basis for accelerated cleavage of bovine pancreatic trypsin inhibitor (BPTI) by human mesotrypsin.J. Biol. Chem. 2008; 283: 4115-4123Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar24.Alloy A.P. Kayode O. Wang R. Hockla A. Soares A.S. Radisky E.S. Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates.J. Biol. Chem. 2015; 290: 21523-21535Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), we have examined relative proteolytic susceptibility of a spectrum of closely related canonical inhibitors belonging to the Kunitz-BPTI 4The abbreviations used are: BPTI, bovine pancreatic trypsin inhibitor; APPI, amyloid precursor protein inhibitor; APLP2-KD, amyloid precursor-like protein 2 Kunitz domain; APPI-3M,APPI-M17G/I18F/F34V triple mutant; MD, molecular dynamics; APLP2-KD*, APLP2-KD cleaved at the Arg-15' Ala-16 reactive site bond; r.m.s.d., root-mean-square deviation; RMSF,root-mean-square fluctuation; MdMD, Maxwell's demon MD; TMD Targeted MD; REX, replica-exchange MD; SMD, steered MD; PDB, Protein Data Bank. 4The abbreviations used are: BPTI, bovine pancreatic trypsin inhibitor; APPI, amyloid precursor protein inhibitor; APLP2-KD, amyloid precursor-like protein 2 Kunitz domain; APPI-3M,APPI-M17G/I18F/F34V triple mutant; MD, molecular dynamics; APLP2-KD*, APLP2-KD cleaved at the Arg-15' Ala-16 reactive site bond; r.m.s.d., root-mean-square deviation; RMSF,root-mean-square fluctuation; MdMD, Maxwell's demon MD; TMD Targeted MD; REX, replica-exchange MD; SMD, steered MD; PDB, Protein Data Bank. family (15.Salameh M.A. Robinson J.L. Navaneetham D. Sinha D. Madden B.J. Walsh P.N. Radisky E.S. The amyloid precursor protein/protease nexin 2 Kunitz inhibitor domain is a highly specific substrate of mesotrypsin.J. Biol. Chem. 2010; 285: 1939-1949Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 25.Salameh M.A. Soares A.S. Navaneetham D. Sinha D. Walsh P.N. Radisky E.S. Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin.J. Biol. Chem. 2010; 285: 36884-36896Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 26.Pendlebury D. Wang R. Henin R.D. Hockla A. Soares A.S. Madden B.J. Kazanov M.D. Radisky E.S. Sequence and conformational specificity in substrate recognition: several human Kunitz protease inhibitor domains are specific substrates of mesotrypsin.J. Biol. Chem. 2014; 289: 32783-32797Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The Kunitz domain, typified by BPTI, possesses a compact pear-shaped protein fold that is stabilized by a hydrophobic core and three conserved disulfide bonds (Fig. 1) (25.Salameh M.A. Soares A.S. Navaneetham D. Sinha D. Walsh P.N. Radisky E.S. Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin.J. Biol. Chem. 2010; 285: 36884-36896Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Surprisingly, despite close conservation of these structural features, different Kunitz family members vary widely in their rates of cleavage by mesotrypsin; human amyloid precursor protein inhibitor (APPI) and amyloid precursor-like protein 2 Kunitz domain (APLP2-KD) are cleaved nearly 3 orders of magnitude faster than BPTI, with substrate-like kinetics (Fig. 1) (15.Salameh M.A. Robinson J.L. Navaneetham D. Sinha D. Madden B.J. Walsh P.N. Radisky E.S. The amyloid precursor protein/protease nexin 2 Kunitz inhibitor domain is a highly specific substrate of mesotrypsin.J. Biol. Chem. 2010; 285: 1939-1949Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 26.Pendlebury D. Wang R. Henin R.D. Hockla A. Soares A.S. Madden B.J. Kazanov M.D. Radisky E.S. Sequence and conformational specificity in substrate recognition: several human Kunitz protease inhibitor domains are specific substrates of mesotrypsin.J. Biol. Chem. 2014; 289: 32783-32797Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Mutagenesis experiments have demonstrated that sequence variation within the binding loops of these inhibitors does not fully account for their differential proteolysis rates; rather, sequence differences in the scaffold, far removed from the enzyme interface, have a profound effect on proteolysis (25.Salameh M.A. Soares A.S. Navaneetham D. Sinha D. Walsh P.N. Radisky E.S. Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin.J. Biol. Chem. 2010; 285: 36884-36896Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Furthermore, in a recent combinatorial protein engineering effort, we identified an APPI variant, APPI-3M, in which only three amino acid substitutions (Fig. 1A) produced a striking ∼100-fold reduction of the proteolysis rate, despite very minimal impact of the mutations on protein structure (Fig. 1B) (27.Cohen I. Kayode O. Hockla A. Sankaran B. Radisky D.C. Radisky E.S. Papo N. Combinatorial protein engineering of proteolytically resistant mesotrypsin inhibitors as candidates for cancer therapy.Biochem. J. 2016; 473: 1329-1341Crossref PubMed Scopus (24) Google Scholar). These differences in catalysis, not easily explained by sequence or static X-ray crystal structures, may be reflective of distinct protein dynamics within the Kunitz domains or at the enzyme interface. In this work, we investigate the structure of the most rapidly cleaved natural Kunitz domain identified to date, APLP2-KD, in complex with mesotrypsin. Unlike prior solved Kunitz domain crystal structures, here APLP2-KD is trapped in a product-like complex, revealing a remarkable conformational transformation after cleavage. This snapshot reveals an unprecedented structural plasticity in the substrate that may be key to its comparatively rapid proteolysis, a hypothesis that we probe through both free (unbiased) and stepwise guided molecular dynamics (MD) simulations. Our results implicate conformational motions within the protein substrate as a determinant of proteolytic rates; this example shows that we must look beyond the enzyme to fully appreciate the importance of protein dynamics in enzyme catalysis. To gain insight into the interaction between rapidly cleaved protein substrate APLP2-KD and mesotrypsin, we cocrystallized the substrate with the catalytically inactive mesotrypsin-S195A variant. The complex crystallized in the P21212 space group with one copy of the dimeric complex in the asymmetric unit, and the structure was solved and refined to 1.4 Å resolution with the statistics described in Table 1. To our surprise, APLP2-KD in this complex displayed hydrolysis of the Arg-15–Ala-16 peptide bond. We will refer to this two-chain form of the substrate as APLP2-KD*, where the asterisk denotes a modified form of APLP2-KD cleaved at the reactive site bond (18.Laskowski Jr., M. Kato I. Protein inhibitors of proteinases.Annu. Rev. Biochem. 1980; 49: 593-626Crossref PubMed Scopus (1940) Google Scholar). Clear electron density revealed the new C terminus of Arg-15, still positioned in the specificity pocket of the enzyme active site (Fig. 2A). However, as the result of a large conformational change in the Kunitz domain, primed side substrate residues 16 and 17 were disordered, whereas residue Met-18, for which only the backbone was visible, was now located >27 Å from Arg-15, near the opposite end of the molecule (Fig. 2B). Continuous electron density was observed throughout the rest of the protein substrate and throughout the mesotrypsin chain. As examination of the active site clearly showed distinct density for the mesotrypsin S195A inactivating mutation (Fig. 2A), it was initially unclear how the substrate reactive site bond had become hydrolyzed. Subsequent analysis of the enzyme preparation used for the crystallographic studies uncovered a small amount (∼0.1%) of enzymatic activity that could be quenched by serine-modifying inhibitor phenylmethylsulfonyl fluoride, revealing minor contamination with nonmutant active mesotrypsin (data not shown). Apparently, the trace amount of active mesotrypsin catalyzed complete limited proteolysis of APLP2-KD at the reactive site; the processed APLP2-KD* then bound to mesotrypsin-S195A, and this product-like complex preferentially crystallized out of solution.TABLE 1X-ray data collection and refinement statisticsMesotrypsin-APLP2-KD* complexPDB code5JBTData collectionSpace groupP21212Cell dimensionsa, b, c (Å)99.01, 54.58, 56.60α, β, γ (°)90, 90, 90Resolution (Å)50–1.40 (1.42–1.40)Rmerge0.064 (0.231)Rmeas0.080 (0.278)Rp.i.m.0.021 (0.102)CC½ND (0.970)I/σI27.2 (6.76)Completeness (%)99.3 (99.8)Redundancy12.8 (7.3)RefinementResolution (Å)50–1.40No. of reflections57,667Rwork/Rfree16.3/19.9No. of atomsProtein2125Ligand/ion5Water259B-factorsProtein19Ligand/ion35Water30Ramachandran statisticsFavored (%)98Allowed (%)2Outliers (%)0r.m.s.d.Bond lengths (Å)0.0219Bond angles (°)2.0874 Open table in a new tab Although subsequent extensive efforts to cocrystallize highly purified mesotrypsin-S195A with intact APLP2-KD were not successful, we are able to gain insight into the APLP2-KD* conformational change through comparisons with the structure of mesotrypsin bound to intact APLP2-KD homolog APPI (Protein Data Bank code 3L33) (25.Salameh M.A. Soares A.S. Navaneetham D. Sinha D. Walsh P.N. Radisky E.S. Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin.J. Biol. Chem. 2010; 285: 36884-36896Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Superposition reveals that although the enzyme is essentially static, the substrate has undergone a massive conformational change upon cleavage, in which the α-helix has rotated nearly 180° to the opposite side of the molecule, and the β-hairpin has become nearly inverted and traverses the center of the Kunitz domain (Fig. 2C). The conformational transformation has little impact on the substrate's nonprimed side residues 11–15, which remain firmly fixed at the enzyme interface with little deviation from their pre-cleavage positions. By contrast, the primed side substrate residues are evacuated from the enzyme active site cleft as a result of the conformational change. Prior studies have suggested that dissociation of primed side residues from the enzyme active site may represent a common rate-limiting step during proteolysis of canonical inhibitors (20.Radisky E.S. Koshland Jr., D.E. A clogged gutter mechanism for protease inhibitors.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10316-10321Crossref PubMed Scopus (130) Google Scholar, 23.Salameh M.A. Soares A.S. Hockla A. Radisky E.S. Structural basis for accelerated cleavage of bovine pancreatic trypsin inhibitor (BPTI) by human mesotrypsin.J. Biol. Chem. 2008; 283: 4115-4123Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 28.Radisky E.S. Kwan G. Karen Lu C.J. Koshland Jr., D.E. Binding, proteolytic, and crystallographic analyses of mutations at the protease-inhibitor interface of the subtilisin BPN′/chymotrypsin inhibitor 2 complex.Biochemistry. 2004; 43: 13648-13656Crossref PubMed Scopus (41) Google Scholar). While acyl-enzyme formation can proceed rapidly, inter- and intramolecular interactions that tether the primed side leaving group can favor peptide bond resynthesis over acyl-enzyme hydrolysis, slowing reaction progress (20.Radisky E.S. Koshland Jr., D.E. A clogged gutter mechanism for protease inhibitors.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10316-10321Crossref PubMed Scopus (130) Google Scholar, 28.Radisky E.S. Kwan G. Karen Lu C.J. Koshland Jr., D.E. Binding, proteolytic, and crystallographic analyses of mutations at the protease-inhibitor interface of the subtilisin BPN′/chymotrypsin inhibitor 2 complex.Biochemistry. 2004; 43: 13648-13656Crossref PubMed Scopus (41) Google Scholar). The motions evidenced by the new crystal structure of cleaved APLP2-KD* appear to facilitate primed side residue dissociation from the enzyme, and thus might help to explain the more rapid proteolysis of this Kunitz domain compared with others. Notably, the only prior reported crystal structure of a post-cleavage Kunitz domain, cleaved BPTI* (where BPTI* is BPTI cleaved at the Lys-15–Ala-16 reactive-site bond) bound to rat anionic trypsin, revealed a native-like structure in which the P1′–P3′ residues were retained in the trypsin active site, poised for peptide bond resynthesis (29.Zakharova E. Horvath M.P. Goldenberg D.P. Structure of a serine protease poised to resynthesize a peptide bond.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 11034-11039Crossref PubMed Scopus (52) Google Scholar). As BPTI is proteolyzed orders of magnitude more slowly than APLP2-KD (Fig. 1), it would appear from these limited data that post-cleavage substrate dynamics may correlate with proteolysis rates. We reasoned that perhaps the conformation observed in the APLP2-KD* complex mimics a productive "open" conformation of the acyl-enzyme, required along the reaction trajectory, which promotes forward reaction progress by allowing water to enter the active site and hydrolyze the acyl-enzyme. Alternatively, the specific conformation stabilized by the crystal lattice may be merely reflective of globally increased substrate motions of APLP2-KD relative to BPTI that may accelerate proteolysis by acting at this or other steps along the reaction pathway. To gain further insight into the potential role of substrate dynamics in governing rates of proteolysis, we next employed free (unbiased) all-atom MD simulations for a series of mesotrypsin-Kunitz domain complexes. The complexes of APPI, APPI-3M, and BPTI with mesotrypsin span a broad range of known catalytic rates (Fig. 1), and for each there was an available experimental X-ray structure of the pre-cleavage Michaelis complex on which to base a starting model (23.Salameh M.A. Soares A.S. Hockla A. Radisky E.S. Structural basis for accelerated cleavage of bovine pancreatic trypsin inhibitor (BPTI) by human mesotrypsin.J. Biol. Chem. 2008; 283: 4115-4123Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 25.Salameh M.A. Soares A.S. Navaneetham D. Sinha D. Walsh P.N. Radisky E.S. Determinants of affinity and proteolytic stability in interactions of Kunitz family protease inhibitors with mesotrypsin.J. Biol. Chem. 2010; 285: 36884-36896Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 27.Cohen I. Kayode O. Hockla A. Sankaran B. Radisky D.C. Radisky E.S. Papo N. Combinatorial protein engineering of proteolytically resistant mesotrypsin inhibitors as candidates for cancer therapy.Biochem. J. 2016; 473: 1329-1341Crossref PubMed Scopus (24) Google Scholar). Because acyl-enzyme hydrolysis and associated conformational changes have been previously postulated to limit catalytic rates (20.Radisky E.S. Koshland Jr., D.E. A clogged gutter mechanism for protease inhibitors.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10316-10321Crossref PubMed Scopus (130) Google Scholar, 23.Salameh M.A. Soares A.S. Hockla A. Radisky E.S. Structural basis for accelerated cleavage of bovine pancreatic trypsin inhibitor (BPTI) by human mesotrypsin.J. Biol. Chem. 2008; 283: 4115-4123Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 28.Radisky E.S. Kwan G. Karen Lu C.J. Koshland Jr., D.E. Binding, proteolytic, and crystallographic analyses of mutations at the protease-inhibitor interface of the subtilisin BPN′/chymotrypsin inhibitor 2 complex.Biochemistry. 2004; 43: 13648-13656Crossref PubMed Scopus (41) Google Scholar), as starting points for simulations we modeled the acyl-enzymes that would be initially formed upon nucleophilic attack by mesotrypsin Ser-195, prior to any significant conformational changes involving primed side residue movement. After energy minimization, the initial positioning of the P1′ residue in all acyl-enzyme models was consistent with the positioning of the P1′ residue of cleaved BPTI* from the previously reported experimental structure (29.Zakharova E. Horvath M.P. Goldenberg D.P. Structure of a serine protease poised to resynthesize a peptide bond.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 11034-11039Crossref PubMed Scopus (52) Google Scholar). Because of the anticipated slow time scale of large substrate motions such as those evidenced in the APLP2-KD* complex, multiple long simulations (1000+ ns/replicate) were conducted for each acyl-enzyme, along with several 100–200-ns replicates for convergence. The results reported for free simulations are based on analyses of an aggregate of >20 μs of trajectory data. To evaluate differences among the complexes in the overall magnitude of global conformational dynamics, we aligned frames on backbone atoms and compared root-mean-square deviation (r.m.s.d.) plots of backbone atom deviation from average positions for all protein residues over the course of the simulations. We observed that the complex involving the more rapidly cleaved APPI substrate sampled a considerably wider range of global conformational states than the complexes with slower proteolyzed substrates APPI-3M or BPTI (Fig. 3A). These differences can be attributed largely to conformational fluctuations of the substrate chain, and comparisons based on r.m.s.d. calculations for the substrate backbone atoms only show yet greater differences in the range of conformational states sampled (Fig. 3B). The per residue root-mean-square fluctuation (RMSF) plots of the individual substrate chains display peak magnitudes that correlate with proteolysis rates observed biochemically; these plots also reveal which residues within each substrate demonstrate the greatest positional deviation over the duration of the simulation (Fig. 3C). By mapping the RMSF values onto the protein structures, we observed that the positional fluctuations of the largest magnitude were evidenced by the α-helix and β-turn regions of the substrate, distal to the point of contact with the enzyme (Fig. 3D). These observations could result from large local conformational fluctuations within these elements of the Kunitz domain secondary structure or, alternatively, from changes in orientation of the substrate at the enzyme interface, where the apparent magnitude of distal fluctuations might be amplified by their greater distance from a pivot point or hinge region. To distinguish between these possibilities, we superposed selected frames from each MD simulation representing a subset of structur
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