The structure of the pro-apoptotic protease granzyme B reveals the molecular determinants of its specificity

Granzyme B is a serine protease of the chymotrypsin fold that mediates cell death by cytotoxic lymphocytes. It is a processing enzyme, requiring extended peptide substrates containing an Asp residue. The determinants that allow for this substrate specificity are revealed in the three-dimensional structure of granzyme B in complex with a macromolecular inhibitor. The primary specificity for Asp occurs through a side-on interaction with Arg 226, a buried Arg side chain of granzyme B. An additional nine amino acids make contact with the substrate and define the granzyme B extended substrate specificity profile. The substrate determinants found in this structure are shared by other members of this protein class and help to reveal the properties that define substrate specificity.     

Residue Asp-189 controls both substrate binding and the monovalent cation specificity of thrombin

Residue Asp-189 plays an important dual role in thrombin: it defines the primary specificity for Arg side chains and participates indirectly in the coordination of Na(+). The former role is shared by other proteases with trypsin-like specificity, whereas the latter is unique to Na(+)-activated proteases in blood coagulation and the complement system. Replacement of Asp-189 with Ala, Asn, Glu, and Ser drastically reduces the specificity toward substrates carrying Arg or Lys at P1, whereas it has little or no effect toward the hydrolysis of substrates carrying Phe at P1. These findings confirm the important role of Asp-189 in substrate recognition by trypsin-like proteases. The substitutions also affect significantly and unexpectedly the monovalent cation specificity of the enzyme. The Ala and Asn mutations abrogate monovalent cation binding, whereas the Ser and Glu mutations change the monovalent cation preference from Na(+) to the smaller cation Li(+) or to the larger cation Rb(+), respectively. The observation that a single amino acid substitution can alter the monovalent cation specificity of thrombin from Na(+) (Asp-189) to Li(+) (Ser-189) or Rb(+) (Glu-189) is unprecedented in the realm of monovalent cation-activated enzymes.     

Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries

Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.     

Analysis of protein processing by N-terminal proteomics reveals novel species-specific substrate determinants of granzyme B orthologs

Using a targeted peptide-centric proteomics approach, we performed in vitro protease substrate profiling of the apoptotic serine protease granzyme B resulting in the delineation of more than 800 cleavage sites in 322 human and 282 mouse substrates, encompassing the known substrates Bid, caspase-7, lupus La protein, and fibrillarin. Triple SILAC (stable isotope labeling by amino acids in cell culture) further permitted intra-experimental evaluation of species-specific variations in substrate selection by the mouse or human granzyme B ortholog. For the first time granzyme B substrate specificities were directly mapped on a proteomic scale and revealed unknown cleavage specificities, uncharacterized extended specificity profiles, and macromolecular determinants in substrate selection that were confirmed by molecular modeling. We further tackled a substrate hunt in an in vivo setup of natural killer cell-mediated cell death confirming in vitro characterized granzyme B cleavages next to several other unique and hitherto unreported proteolytic events in target cells.     

Electrostatic reversal of serine proteinase substrate specificity.

Mouse granzyme B is a member of the chymotrypsin family of serine proteinases that has an unusual preference for cleavage of substrates following aspartate residues. We show here that granzyme B can be redesigned by a single amino acid substitution in one wall of the specificity pocket, arginine-226 to glutamate, to hydrolyze preferentially thioester substrates following basic amino acids. Amide substrates, however, were not hydrolyzed by the variant granzyme B. These results show that residue 226 is a primary determinant of granzyme B specificity and imply that additional structural components are required for catalysis of amide bonds. Molecular modeling indicated subtle variation in glutamate-226 orientation depending upon the state of protonation of the gamma-carboxylate, which may account for the secondary specificity of this enzyme for substrates containing phenylalanine. This represents the first example of electrostatic reversal of serine proteinase substrate specificity and suggests that residue 226 is a primary substrate specificity determinant in the granzyme B lineage of serine proteinases.     

Distinct contributions of residue 192 to the specificity of coagulation and fibrinolytic serine proteases

Archetypal members of the chymotrypsin family of serine proteases, such as trypsin, chymotrypsin, and elastase, exhibit relatively broad substrate specificity. However, the successful development of efficient proteolytic cascades, such as the blood coagulation and fibrinolytic systems, required the evolution of proteases that displayed restricted specificity. Tissue-type plasminogen activator (t-PA), for example, possesses exquisitely stringent substrate specificity, and the molecular basis of this important biochemical property of t-PA remains obscure. Previous investigations of related serine proteases, which participate in the blood coagulation cascade, have focused attention on the residue that occupies position 192 (chymotrypsin numbering system), which plays a pivotal role in determining both the inhibitor and substrate specificity of these enzymes. Consequently, we created and characterized the kinetic properties of new variants of t-PA that contained point mutations at position 192. These studies demonstrated that, unlike in coagulation serine proteases, Gln-192 does not contribute significantly to the substrate or inhibitor specificity of t-PA in physiologically relevant reactions. Replacement of Gln-192 with a glutamic acid residue did, however, decrease the catalytic efficiency of mature, two-chain t-PA toward plasminogen in the absence of a fibrin co-factor.     

Extended substrate specificity of rat mast cell protease 5, a rodent alpha-chymase with elastase-like primary specificity

Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of alpha-chymases from different species indicated that certain rodent alpha-chymases have a restricted S1 pocket that could only accommodate small amino acids, i.e. they may, despite being classified as chymases, in fact display elastase-like substrate specificity. To explore this possibility, the alpha-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His6 purification tag and an enterokinase-susceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serine-protease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-Ala-Ala-Pro-X-p-nitroanilide, where the X was Ile, Val, Ala, Phe or Leu. The activity was highest toward substrates with Val or Ala in the P1 position, whereas low activity toward the peptide with a P1 Phe was observed, indicating that the substrate specificity of rMCP-5 indeed is elastase-like. The extended substrate specificity was examined utilizing a phage-displayed random nonapeptide library. The preferred cleavage sequence was resolved as P4-(Gly/Pro/Val), P3-(Leu/Val/Glu), P2-(Leu/Val/Thr), P1-(Val/Ala/Ile), P1'-(Xaa), and P2'-(Glu/Leu/Asp). Hence, the extended substrate specificity is similar to human chymase in most positions except for the P1 position. We conclude that the rat alpha-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human alpha-chymase.     

A missense mutation at Ile172----Asn or Arg356----Trp causes steroid 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a common recessive genetic disease caused mainly by steroid 21-hydroxylase (P450c21) deficiency. Many forms of CAH exist resulting from various mutations of the CYP21B gene. We sequenced CYP21B cDNA from a normal person and its genes from a patient with simple virilizing CAH. When comparing several CYP21B sequences, we found it was polymorphic. In the patient, a single base substitution replaced Ile172 (ATC) with Asn (AAC) in one allele while Arg356 (CGG) was converted to Trp (TGG) in the other. A normal P450c21 cDNA clone was transfected into COS-1 cells to produce 21-hydroxylase activity toward its substrates, progesterone and 17-hydroxyprogesterone. Mutants corresponding to Asn172 or Trp356 mutation were constructed by site-directed mutagenesis of the normal c21 cDNA clone. They failed to produce active enzyme toward either substrate upon transfection into COS-1 cells, demonstrating that these mutations caused CAH. Aligning sequences with other P450s, Ile172 could be located in the membrane anchoring domain and Arg356 in the substrate-binding site of P450c21. Both mutations are present in the CYP21A1P pseudogene, suggesting that they may be transferred from CYP21A1P by gene conversion events.     

Mutations in Arg143 and Lys192 of the Human Mast Cell Chymase Markedly Affect the Activity of Five Potent Human Chymase Inhibitors

Chymotrypsin-like serine proteases are found in high abundance in mast cell granules. By site-directed mutatgenesis, we have previously shown that basic amino acids in positions 143 and 192 (Arg and Lys respectively) of the human mast cell chymase are responsible for an acidic amino acid residue preference in the P2'' position of substrates. In order to study the influence of these two residues in determining the specificity of chymase inhibitors, we have synthesized five different potent inhibitors of the human chymase. The inhibitory effects of these compounds were tested against the wild-type enzyme, against two single mutants Arg143Gln and Lys192Met and against a double mutant, Arg143Gln+Lys192Met. We observed a markedly reduced activity of all five inhibitors with the double mutant, indicating that these two basic residues are involved in conferring the specificity of these inhibitors. The single mutants showed an intermediate phenotype, with the strongest effect on the inhibitor by the mutation in Lys192. The Lys192 and the double mutations also affected the rate of cleavage of angiotensin I but did not seem to affect the specificity in the cleavage of the Tyr₄-Ile₅ bond. A more detailed knowledge about which amino acids that confer the specificity of an enzyme can prove to be of major importance for development of highly specific inhibitors for the human chymase and other medically important enzymes.     

A simple method for the determination of individual rate constants for substrate hydrolysis by serine proteases

A simple method is presented for the determination of individual rate constants for substrate hydrolysis by serine proteases and other enzymes with similar catalytic mechanism. The method does not require solvent perturbation like viscosity changes, or solvent isotope effects, that often compromise nonspecifically the activity of substrate and enzyme. The rates of substrate diffusion into the active site (k1), substrate dissociation (k-1), acylation (k2), and deacylation (k3) in the accepted mechanism of substrate hydrolysis by serine proteases are derived from the temperature dependence of the Michaelis-Menten parameters kcat/Km and kcat. The method also yields the activation energies for these molecular events. Application to wild-type and mutant thrombins reveals how the various steps of the catalytic mechanism are affected by Na+-binding and site-directed mutations of the important residues Y225 in the Na+ binding environment and L99 in the S2 specificity site. Extension of this method to other proteases should enable the derivation of detailed information on the kinetic and energetic determinants of protease function.     

Mouse and human granzyme B have distinct tetrapeptide specificities and abilities to recruit the bid pathway

Granzyme B is an important mediator of cytotoxic lymphocyte granule-induced death of target cells, accomplishing this through cleavage of Bid and cleavage and activation of caspases as well as direct cleavage of downstream substrates. Significant controversy exists regarding the primary pathways used by granzyme B to induce cell death, perhaps arising from the use of different protease/substrate combinations in different studies. The primary sequence of human, rat, and mouse granzymes B is well conserved, and the substrate specificity and crystal structure of the human and rat proteases are extremely similar. Although little is known about the substrate specificity of mouse granzyme B, recent studies suggest that it may differ significantly from the human protease. In these studies we show that the specificities of human and mouse granzymes B differ significantly. Human and mouse granzyme B cleave species-specific procaspase-3 more efficiently than the unmatched substrates. The distinct specificities of human and mouse granzyme B highlight a previously unappreciated requirement for Asp(192) in the acquisition of catalytic activity upon cleavage of procaspase-3 at Asp(175). Although human granzyme B efficiently cleaves human or mouse Bid, these substrates are highly resistant to cleavage by the mouse protease, strongly indicating that the Bid pathway is not a major primary mediator of the effects of mouse granzyme B. These studies provide important insights into the substrate specificity and function of the granzyme B pathway in different species and highlight that caution is essential when designing and interpreting experiments with different forms of granzyme B.     

Multiple pocket recognition of SNAP25 by botulinum neurotoxin serotype E

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. There are seven serotypes of BoNT, termed A-G. The molecular basis for SNAP25 recognition and cleavage by BoNT serotype E is currently unclear. Here we define the multiple pocket recognition of SNAP25 by LC/E. The initial recognition of SNAP25 is mediated by the binding of the B region of SNAP25 to the substrate-binding (B) region of LC/E comprising Leu166, Arg167, Asp127, Ala128, Ser129, and Ala130. The mutations at these residues affected substrate binding and catalysis. Three additional residues participate in scissile bond cleavage of SNAP25 by LC/E. The P3 site residues, Ile178, of SNAP25 interacted with the S3 pocket in LC/E through hydrophobic interactions. The S3 pocket included Ile47, Ile164, and Ile182 and appeared to align the P1' and P2 residues of SNAP25 with the S1' and S2 pockets of LC/E. The S1' pocket of LC/E included three residues, Phe191, Thr159, and Thr208, which contribute hydrophobic and steric interactions with the SNAP25 P1' residue Ile181. The S2 pocket residue of LC/E, Lys224, binds the P2 residue of SNAP25, Asp179, through ionic interactions. Deletion mapping indicates that main chain interaction(s) of residues 182-186 of SNAP25 contribute to substrate recognition by LC/E. Understanding the mechanism for substrate specificity provides insight for the development of inhibitors against the botulinum neurotoxins.     

Granzyme M is a regulatory protease that inactivates proteinase inhibitor 9, an endogenous inhibitor of granzyme B

Granzyme M is a trypsin-fold serine protease that is specifically found in the granules of natural killer cells. This enzyme has been implicated recently in the induction of target cell death by cytotoxic lymphocytes, but unlike granzymes A and B, the molecular mechanism of action of granzyme M is unknown. We have characterized the extended substrate specificity of human granzyme M by using purified recombinant enzyme, several positional scanning libraries of coumarin substrates, and a panel of individual p-nitroanilide and coumarin substrates. In contrast to previous studies conducted using thiobenzyl ester substrates (Smyth, M. J., O'Connor, M. D., Trapani, J. A., Kershaw, M. H., and Brinkworth, R. I. (1996) J. Immunol. 156, 4174-4181), a strong preference for leucine at P1 over methionine was demonstrated. The extended substrate specificity was determined to be lysine = norleucine at P4, broad at P3, proline > alanine at P2, and leucine > norleucine > methionine at P1. The enzyme activity was found to be highly dependent on the length and sequence of substrates, indicative of a regulatory function for human granzyme M. Finally, the interaction between granzyme M and the serpins alpha(1)-antichymotrypsin, alpha(1)-proteinase inhibitor, and proteinase inhibitor 9 was characterized by using a candidate-based approach to identify potential endogenous inhibitors. Proteinase inhibitor 9 was effectively hydrolyzed and inactivated by human granzyme M, raising the possibility that this orphan granzyme bypasses proteinase inhibitor 9 inhibition of granzyme B.     

The factor V-activating enzyme (RVV-V) from Russell's viper venom. Identification of isoproteins RVV-V alpha, -V beta, and -V gamma and their complete amino acid sequences

The complete amino acid sequences of two isoproteins of the factor V-activating enzyme (RVV-V) isolated from Vipera russelli (Russell's viper) venom were determined by sequencing S-pyridylethylated derivatives of the proteins and their peptide fragments generated by either chemical (cyanogen bromide and 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine) or enzymatic (trypsin, alpha-chymotrypsin, and lysyl endopeptidase) cleavages. Both enzymes, designated RVV-V alpha and RVV-V gamma, consist of 236 amino acid residues and have a N-linked oligosaccharide chain at Asn229. The six amino acid substitutions between RVV-V alpha and -V gamma are: Thr22(alpha)-Ala22(gamma), Gly29(alpha)-Ala29(gamma), Gln191(alpha)-Glu191(gamma), Ile192(alpha)-Met192(gamma), Gln193(alpha)-His193(gamma), and Asn224(alpha)-Ser224(gamma). The molecular weights were calculated as 26,182 for RVV-V alpha and 26,167 for RVV-V gamma. The sequences of the RVV-V isoproteins exhibited 62% identity with that of batroxobin, a thrombin-like enzyme present in Bothrops atrox venom, and 33% identity with that of human thrombin B chain. The most interesting difference between the structures of RVV-V and other trypsin-type serine proteases is that the conservative Ser214-Trp215-Gly216 sequence (chymotrypsinogen numbering), considered as the site of antiparallel beta-sheet formation between the protein substrate and most serine proteases, has been replaced by the corresponding sequence Ala-Gly-Gly.     

Evidence for the Existence of Granzyme-Like Serine Proteases in Teleost Cytotoxic Cells

Granzymes are granule-associated serine proteases, which are important effector molecules in NK cell and CTL functions. The granzyme family poses a perplexing problem in phylogenetics due to the lack of nonmammalian sequence information. We now report the identification of a cDNA that codes for a granzyme homologue, channel catfish granzyme-1 (CFGR-1), from nonspecific cytotoxic cells (NCC) of a teleost. NCC are the first identified and extensively studied cytotoxic cell population in teleosts. Ictalurus punctatus (channel catfish) granzyme cDNA encodes a protein with ~50% similarity to granzymes A and K. Highly conserved catalytic triad residues of serine proteases and other motifs common to granzymes were also identified. Conserved amino acid sequences, structure–function data available for the serine protease family, and the crystal structure of human granzyme K supported a model of CFGR-1. It suggested an Arg/Lys primary substrate specificity that is shared with granzymes A and K. Furthermore, CFGR-1 has the four conserved disulfide bonds of granzymes A, K, and M. Phylogenetic analysis suggested that this molecule is a member of the granzyme family. Expression of CFGR-1 in NCC was confirmed by RT-PCR analysis. Presence of a granzyme-like molecule that might play an important role in the effector functions of NCC indicates that cell-mediated immunity with granule exocytosis and Fas pathways have been conserved for more than 300 million years.     

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.     

cDNA cloning of granzyme C, a granule-associated serine protease of cytolytic T lymphocytes

A cDNA clone corresponding to the complete amino acid sequence of a putative protease CCP2 of murine cytotoxic T lymphocytes was isolated and sequenced. The clone encodes a 248-residue long serine esterase. The deduced N-terminal amino acid sequence is identical over 40 residues to that of granzyme C, a protease of unknown function present in granules of cytotoxic lymphocytes. Analysis of the sequence of granzyme C/CCP2 reveals high homology to other granzyme proteases, i.e. granzyme A (40%) and granzyme B (67%) and to rat mast cell protease II (46%). The amino acids lining the specificity pocket are well conserved between granzyme B, C, and rat mast cell protease II, but not granzyme A, suggesting a similar general specificity of these three proteases.     

Conservation of the Extended Substrate Specificity Profiles Among Homologous Granzymes Across Species

Granzymes are structurally related serine proteases involved in cell death and immunity. To date four out of five human granzymes have assigned orthologs in mice; however for granzyme H, no murine ortholog has been suggested and its role in cytotoxicity remains controversial. Here, we demonstrate that, as is the case for granzyme C, human granzyme H is an inefficient cytotoxin that together with their similar pattern of GrB divergence and functional similarity strongly hint to their orthologous relationship. Besides analyzing the substrate specificity profile of granzyme H by substrate phage display, substrate cleavage susceptibility of human granzyme H and mouse granzyme C was assessed on a proteome-wide level. The extended specificity profiles of granzymes C and H (i.e. beyond cleavage positions P4-P4′) match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes as granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can simply be conferred by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility.Several molecular mechanisms are in place to combat transformed malignant cells and virally infected cells. Granzymes (Gr)¹, a family of structurally related serine proteases found in the granules of many immune cells, play crucial roles in such cellular defense mechanisms. The granzyme family consists of five human proteases (granzymes A, B, H, K, and M) and 10 murine members (granzymes A to G, K, M, and N). To date for four human granzymes (A, B, K, and M) clear murine orthologs have been assigned, while the most probable murine ortholog of human granzyme H (GrH) is granzyme C (GrC) based on their 70% sequence similarity, 61% sequence identity, and identical chromosomal location relative to granzyme B (GrB). Furthermore, both granzymes are expressed by NK and CD4+ T-cells (1, 2): GrH is constitutively expressed at high levels in NK cells and less in CD4+ T cells, whereas GrC expression can be observed after stimulation. Thus, their overlapping expression profiles further support possible functional similarities between mouse GrC and human GrH.The physiological role of granzymes was presumed to be the induction of death in target cells. Granzyme B is a highly efficient cytotoxin (3) and other granzymes such as GrA, GrC, GrF, and GrK can cause cell death at high concentrations (4–8). Recently, granzymes A, K, and M were shown to steer inflammatory processes when used at physiological levels (9–11). Two previous studies identified GrH as an alternative cytotoxic effector protease. Although both studies showed typical hallmarks of apoptosis, including mitochondrial depolarization, reactive oxygen species (ROS) production, DNA degradation as well as chromatin condensation, Fellows et al. (12) found that, in contrast to GrB mediated cell death, GrH induced cell death did not result in caspase activation, cytochrome c release, or cleavage of Bid and/or ICAD. In sharp contrast however, Hou et al. (13) demonstrated that GrH induced apoptosis depended on caspase activation and that GrH cleaved ICAD and Bid, the latter ultimately resulting in mitochondrial cytochrome c release. Such discrepancies have been documented in other granzyme studies and are usually linked to the use of different granzyme delivery systems, sources of recombinantly produced granzymes, differences in the granzyme concentration and species-specific differences in substrate specificities (14).GrC induces cell death reminiscent of GrH induced cell death as observed by Fellows et al. (12), as both exert their cytotoxic functions independent of caspase activation, Bid or ICAD cleavage, or by mitochondrial release of cytochrome c (5). GrC induced apoptosis was characterized by the rapid externalization of phosphatidylserine, nuclear condensation and collapse, and single-stranded DNA nicking. Supporting evidence implying a role for GrC in lymphocyte induced cytotoxicity was inferred from the fact that GrB cluster-deficient mice (mice that do not express GrB and show a five- to sixfold reduced expression of GrC and GrF respectively) display a more pronounced defect in the clearance of allogeneic tumor cells when compared with GrB-only knockout mice (4). This suggests that GrC and/or GrF may be important for correct functioning of cytotoxic lymphocytes. Besides, in GrB-only knockout mice, a likely compensatory mechanism occurs, given that during cytotoxic lymphocyte activation, peak expression of GrC occurs earlier, giving rise to overall higher GrC levels as compared with wild-type mice (1, 4, 15). Of note, despite its implication in cytotoxicity, GrC was shown to be an inefficient cytotoxin, with a 2900-fold greater EC₅₀ value as compared with hGrB when delivered into P815 cells with recombinant mouse perforin (16).Positional Scanning Synthetic Combinatorial Libraries (PS-SCL) revealed the chymotrypsin-like activity of GrH, which it shares with granzyme M (17–19), with an optimal P4-P1 peptide substrate sequence Pro-Thr-Ser-Tyr. Less stringent specificities were observed at positions P4, P3, and P2 (19) where GrH seems to tolerate multiple amino acids with different chemical characteristics (especially neutral and aliphatic amino acids). Although GrH and GrM (optimal peptide identified as Lys-Val-Pro-Leu) share a P1 chymotryptic activity, GrH prefers bulkier, aromatic amino acids (Tyr and Phe) at P1 whereas GrM prefers Leu. Both further recognize Leu and Met at P1, implying that some substrates could be cleaved by both granzymes. GrM also shows broader specificities at P3, but at P4 and P2 it prefers basic residues and Pro respectively. Besides, GrC chymase activity could be inferred from N-terminal COFRADIC and substrate phage display screens, which defined the P4-P3′ substrate specificity of GrC as [Ile/Val]-X-[Phe-Tyr]-[Phe-Leu-Tyr-Met]↓X-[Gly-Ser]-[Asp-Glu] (16).Recently, the crystal structure of the D₁₀₂>N GrH variant in complex with a decapeptide substrate (PTSYAGDDSG) or inhibitor (Ac-PTSY-chloromethylketone) was resolved (20). The electron density maps clearly showed the full length protease adopting a canonical structure of 2 α-helices and 13 β-strands assembled into two juxtaposed β-barrel domains bridged by the catalytic triad (composed of His₅₇, Asp₁₀₂ and Ser₁₉₅). The S1 specificity pocket is built up from residues of 2 loops; loop 189 (from residue 183–196) and loop 220 (from residue 215–226) with the determinants being Thr₁₈₉, Gly₂₁₆, and Gly₂₂₆. From these, Gly₂₂₆ was assigned as the most important determinant for the preference of bulky aromatic amino acids (such as Tyr and Phe) at P1. Where GrH contains a Gly at position 226, GrB, GrC, and GrM harbor an Arg, Gln, and Pro residue respectively (supplemental Fig. S1). Mutation of Gly₂₂₆ in GrH to Arg₂₂₆ compromised binding of bulky aromatic residues and enabled interaction with negatively charged amino acids. Hydrogen bond formation between a P1 Tyr and Asn₂₁₇ of GrH could furthermore strengthen the observed preference of Tyr over Phe at P1 (19). The presence of Pro at position 226 in GrM instead of Gly narrows the S1 pocket and might be indicative for the preference of Leu instead of Phe and Tyr at P1. In addition, the structure revealed that the S4′ pocket formed by the backbones of the Arg₃₉-Lys₄₀-Arg₄₁ motif resulted in a preference for acidic residues at P4′, in addition to influencing the P3′ preference for acidic residues via salt bridge formation with this Lys₄₀. Although the Arg₃₉-Lys₄₀-Arg₄₁ motif is a unique GrH feature, GrB possesses a partially degenerate Leu-Lys-Arg motif in which Lys₄₀ also enables interaction with acidic residues in P3′. Next to P3′, proteome-wide screening for GrB substrates led to the identification of a clear preference for acidic residues at P4′ caused by salt bridge formation with Arg₄₁ (21), a characteristic that was previously assigned to Lys₄₀ of GrB (22). To validate these structural observations, similar to the P4′ Asp mutation in the GrB substrate PI-9 (22), both Asp residues found at P3′ and P4′ in the nuclear phosphoprotein La, previously identified as a macromolecular GrH substrate (23), were mutated to Ala. These mutations completely abolished GrH mediated proteolysis, a first indication that the P4-P1 specificity profile is not a sole determinant for substrate recognition by GrH. Next to the La phosphoprotein, cleavage of the viral adenovirus DNA binding protein (DBP) and the 100K assembly protein (L4–100K), the latter resulting in the relieve of GrB inhibition by L4–100K, has been observed, implicating GrH in host antiviral defense and indicative for a functional synergism between GrH and GrB (24).Elucidation of the crystal structure, with an electron density map showing residues Ile₁₆-His₂₄₄ (i.e. 94% of full length GrC and 99.6% of active GrC), furthermore showed that wild-type GrC can be restrained in its proteolytic function despite the presence of the catalytic triad residues His₅₇-Asp₁₀₂-Ser₁₉₅ (16). Comparing the crystal structures of GrC and GrA showed an unusual conformation of the active site, which could explain the inactivity of GrC. Apparently, the 190-strand, preceding Ser₁₉₅ of the catalytic triad, has undergone a register shift on the structural level leading to Phe₁₉₁ filling the S1 pocket. This pocket is furthermore covered by Glu₁₉₂, which forms a salt bridge with Arg₉₉ and a hydrogen bond with the backbone amide of Ser₁₉₅. Because of this unusual conformation, the Glu₁₉₂-Glu₁₉₃ peptide bond points away from the substrate, as such leading to an improperly formed oxyanion hole, which normally stabilizes the negatively charged substrate oxygen atom during catalysis. Mutation of Glu₁₉₂-Glu₁₉₃ to the corresponding amino acids in its closest related homolog GrB (Arg₁₉₂-Gly₁₉₃) disrupted the Glu₁₉₂-Ser₁₉₅ hydrogen bond and caused a shift of the 190-strand, thereby clearing the S1 pocket and giving rise to an active GrC mutant. These results indicate allosteric control of wild-type GrC in which binding of a substrate or cofactor might stabilize the 190-strand, which becomes extremely mobile due to breaking the Glu₁₉₂-Ser₁₉₅ hydrogen bond and turning the region surrounding the active site more rigid.To elucidate a possible functional homology between GrH and GrC, we performed differential degradome analyses using N-terminal COFRADIC in the species-matching proteome backgrounds. For these analyses, we made use of the active E₁₉₂E₁₉₃>RG GrC mutant as described in (16) (further referred to as mut GrC). These analyses, further complemented with phage display data on granzyme H, clearly show that both granzymes display a highly similar substrate specificity profile, analogous to other orthologous granzymes. Besides, and in contrast to GrA, a general conservation of the extended substrate specificity profiles among the homologous granzymes B, C, H, and M could be observed across species, highlighting the importance of the extended substrate specificity in steering substrate cleavage susceptibility.     

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.     

Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes

A human CD8+ CTL clone with cytolytic potential was shown to express two serine proteases, a 50-kDa homodimer and a 27-kDa monomer, which were purified from cytoplasmic granules. N-terminal sequencing of the purified proteins revealed that the 50-kDa homodimer is the gene product of the human Hanukah factor cDNA clone and that it represents the human homologue to granzyme A. Similarly, the 27-kDa protein was shown to be the serine esterase encoded by the human lymphocyte protease cDNA clone and corresponds to granzyme B. There was no evidence for the presence of other granzymes, in particular for the human homologues to murine granzymes C, D, E, and F. The substrate best cleaved by granzyme A was Gly-Pro-Arg-amido-4-methyl-coumarin after the Arg residue and the pH optimum was 8 to 8.5. Upon triggering of the TCR-CD3 complex with an anti-CD3 mAb, granzyme A was released into the culture medium. Furthermore, a granule-associated hemolytic activity was detected after salt extraction and partial purification of granule proteins. This suggests that hemolytically active human perforin can be obtained from inactive granules.