Disulfide engineering of human Kunitz-type serine protease inhibitors enhances proteolytic stability and target affinity toward mesotrypsin.
ABSTRACT: Serine protease inhibitors of the Kunitz-bovine pancreatic trypsin inhibitor (BPTI) family are ubiquitous biological regulators of proteolysis. These small proteins are resistant to proteolysis, but can be slowly cleaved within the protease-binding loop by target proteases, thereby compromising their activity. For the human protease mesotrypsin, this cleavage is especially rapid. Here, we aimed to stabilize the Kunitz domain structure against proteolysis through disulfide engineering. Substitution within the Kunitz inhibitor domain of the amyloid precursor protein (APPI) that incorporated a new disulfide bond between residues 17 and 34 reduced proteolysis by mesotrypsin 74-fold. Similar disulfide engineering of tissue factor pathway inhibitor-1 Kunitz domain 1 (KD1TFPI1) and bikunin Kunitz domain 2 (KD2bikunin) likewise stabilized these inhibitors against mesotrypsin proteolysis 17- and 6.6-fold, respectively. Crystal structures of disulfide-engineered APPI and KD1TFPI1 variants in a complex with mesotrypsin at 1.5 and 2.0 Å resolution, respectively, confirmed the formation of well-ordered disulfide bonds positioned to stabilize the binding loop. Long all-atom molecular dynamics simulations of disulfide-engineered Kunitz domains and their complexes with mesotrypsin revealed conformational stabilization of the primed side of the inhibitor-binding loop by the engineered disulfide, along with global suppression of conformational dynamics in the Kunitz domain. Our findings suggest that the Cys-17-Cys-34 disulfide slows proteolysis by dampening conformational fluctuations in the binding loop and minimizing motion at the enzyme-inhibitor interface. The generalizable approach developed here for the stabilization against proteolysis of Kunitz domains, which can serve as important scaffolds for therapeutics, may thus find applications in drug development.
Project description:An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P(1) (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'(2) favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P(1) and P'(2) substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin·APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.
Project description:Mesotrypsin is an isoform of trypsin that is uniquely resistant to polypeptide trypsin inhibitors and can cleave some inhibitors rapidly. Previous studies have shown that the amyloid precursor protein Kunitz protease inhibitor domain (APPI) is a specific substrate of mesotrypsin and that stabilization of the APPI cleavage site in a canonical conformation contributes to recognition by mesotrypsin. We hypothesized that other proteins possessing potential cleavage sites stabilized in a similar conformation might also be mesotrypsin substrates. Here we evaluated a series of candidate substrates, including human Kunitz protease inhibitor domains from amyloid precursor-like protein 2 (APLP2), bikunin, hepatocyte growth factor activator inhibitor type 2 (HAI2), tissue factor pathway inhibitor-1 (TFPI1), and tissue factor pathway inhibitor-2 (TFPI2), as well as E-selectin, an unrelated protein possessing a potential cleavage site displaying canonical conformation. We find that Kunitz domains within APLP2, bikunin, and HAI2 are cleaved by mesotrypsin with kinetic profiles of specific substrates. TFPI1 and TFPI2 Kunitz domains are cleaved less efficiently by mesotrypsin, and E-selectin is not cleaved at the anticipated site. Cocrystal structures of mesotrypsin with HAI2 and bikunin Kunitz domains reveal the mode of mesotrypsin interaction with its canonical substrates. Our data suggest that major determinants of mesotrypsin substrate specificity include sequence preferences at the P1 and P'2 positions along with conformational stabilization of the cleavage site in the canonical conformation. Mesotrypsin up-regulation has been implicated previously in cancer progression, and proteolytic clearance of Kunitz protease inhibitors offers potential mechanisms by which mesotrypsin may mediate pathological effects in cancer.
Project description:Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.
Project description:PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumour progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P(2)' position. We find that bulky and charged residues strongly disfavour binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P(1) and P(2)' residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant K(i) of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of the present study support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.
Project description:Engineered protein therapeutics offer advantages, including strong target affinity, selectivity and low toxicity, but like natural proteins can be susceptible to proteolytic degradation, thereby limiting their effectiveness. A compelling therapeutic target is mesotrypsin, a protease up-regulated with tumour progression, associated with poor prognosis, and implicated in tumour growth and progression of many cancers. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biological inhibitors. We used a powerful yeast display platform for directed evolution, employing a novel multi-modal library screening strategy, to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity and improved selectivity for mesotrypsin inhibition. We identified a triple mutant APPIM17G/I18F/F34V, with a mesotrypsin inhibition constant (Ki) of 89 pM, as the strongest mesotrypsin inhibitor yet reported; this variant displays 1459-fold improved affinity, up to 350?000-fold greater specificity and 83-fold improved proteolytic stability compared with wild-type APPI. We demonstrated that APPIM17G/I18F/F34V acts as a functional inhibitor in cell-based models of mesotrypsin-dependent prostate cancer cellular invasiveness. Additionally, by solving the crystal structure of the APPIM17G/I18F/F34V-mesotrypsin complex, we obtained new insights into the structural and mechanistic basis for improved binding and proteolytic resistance. Our study identifies a promising mesotrypsin inhibitor as a starting point for development of anticancer protein therapeutics and establishes proof-of-principle for a novel library screening approach that will be widely applicable for simultaneously evolving proteolytic stability in tandem with desired functionality for diverse protein scaffolds.
Project description:High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid ?-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and kallikrein-6. We generated a variant, designated APPIP13W/M17G/I18F/F34V, with up to 30-fold greater specificity relative to the parental APPIM17G/I18F/F34V protein, and 6500- to 230?000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by the interaction of the mutated APPI residues with nonconserved enzyme residues located in or near the binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins.
Project description:Mesotrypsin displays unusual resistance to inhibition by polypeptide trypsin inhibitors and cleaves some such inhibitors as substrates, despite a high degree of conservation with other mammalian trypsins. Substitution of Arg for the generally conserved Gly-193 has been implicated as a critical determinant of the unusual behavior of mesotrypsin toward protein protease inhibitors. Another relatively conserved residue near the trypsin active site, Tyr-39, is substituted by Ser-39 in mesotrypsin. Tyr-39, but not Ser-39, forms a hydrogen bond with the main chain amide nitrogen of the P(4) ' residue of a bound protease inhibitor. To investigate the role of the Tyr-39 H-bond in trypsin-inhibitor interactions, we reciprocally mutated position 39 in mesotrypsin and human cationic trypsin to Tyr-39 and Ser-39, respectively. We assessed inhibition constants and cleavage rates of canonical protease inhibitors bovine pancreatic trypsin inhibitor (BPTI) and the amyloid precursor protein Kunitz protease inhibitor domain by mesotrypsin and cationic trypsin variants, finding that the presence of Ser-39 relative to Tyr-39 results in a 4- to 13-fold poorer binding affinity and a 2- to 18-fold increase in cleavage rate. We also report the crystal structure of the mesotrypsin-S39Y•BPTI complex, in which we observe an H-bond between Tyr-39 OH and BPTI Ile-19 N. Our results indicate that the presence of Ser-39 in mesotrypsin, and corresponding absence of a single H-bond to the inhibitor backbone, makes a small but significant functional contribution to the resistance of mesotrypsin to inhibition and the ability of mesotrypsin to proteolyze inhibitors.
Project description: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.
Project description:Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity promotes cancer invasion and metastasis. This characteristic suggests KLK6 as an attractive target for therapeutic interventions. However, inhibitors that specifically target KLK6 have not yet been reported, possibly because KLK6 shares a high sequence homology and structural similarity with other serine proteases and resists inhibition by many polypeptide inhibitors. Here, we present an innovative combinatorial approach to engineering KLK6 inhibitors via flow cytometry-based screening of a yeast-displayed mutant library of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI), an inhibitor of other serine proteases, such as anionic and cationic trypsins. On the basis of this screening, we generated APPIM17L,I18F,S19F,F34V (APPI-4M), an APPI variant with a KLK6 inhibition constant (Ki ) of 160 pm and a turnover time of 10 days. To the best of our knowledge, APPI-4M is the most potent KLK6 inhibitor reported to date, displaying 146-fold improved affinity and 13-fold improved proteolytic stability compared with WT APPI (APPIWT). We further demonstrate that APPI-4M acts as a functional inhibitor in a cell-based model of KLK6-dependent breast cancer invasion. Finally, the crystal structures of the APPIWT/KLK6 and APPI-4M/KLK6 complexes revealed the structural and mechanistic bases for the improved KLK6 binding and proteolytic resistance of APPI-4M. We anticipate that APPI-4M will have substantial translational potential as both imaging agent and therapeutic.
Project description:Mesotrypsin is an unusual human trypsin isoform with inhibitor resistance and the ability to degrade trypsin inhibitors. Degradation of the protective serine protease inhibitor Kazal type 1 (SPINK1) by mesotrypsin in the pancreas may contribute to the pathogenesis of pancreatitis. Here we tested the hypothesis that the regulatory digestive protease chymotrypsin C (CTRC) mitigates the harmful effects of mesotrypsin by cleaving the autolysis loop. As human trypsins are post-translationally sulfated in the autolysis loop, we also assessed the effect of this modification. We found that mesotrypsin cleaved in the autolysis loop by CTRC exhibited catalytic impairment on short peptides due to a 10-fold increase in <i>K<sub>m</sub></i> , it digested ?-casein poorly and bound soybean trypsin inhibitor with 10-fold decreased affinity. Importantly, CTRC-cleaved mesotrypsin degraded SPINK1 with markedly reduced efficiency. Sulfation increased mesotrypsin activity but accelerated CTRC-mediated cleavage of the autolysis loop and did not protect against the detrimental effect of CTRC cleavage. The observations indicate that CTRC-mediated cleavage of the autolysis loop in mesotrypsin decreases protease activity and thereby protects the pancreas against unwanted SPINK1 degradation. The findings expand the role of CTRC as a key defense mechanism against pancreatitis through regulation of intrapancreatic trypsin activity.