Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137, 180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.     

Protease specificity determination by using cellular libraries of peptide substrates (CLiPS)

We report a general combinatorial approach to identify optimal substrates of a given protease by using quantitative kinetic screening of cellular libraries of peptide substrates (CLiPS). A whole-cell protease activity assay was developed by displaying fluorescent reporter substrates on the surface of Escherichia coli as N-terminal fusions. This approach enabled generation of substrate libraries of arbitrary amino acid composition and length that are self-renewing. Substrate hydrolysis by a target protease was measured quantitatively via changes in whole-cell fluorescence by using FACS. FACS enabled efficient screening to identify optimal substrates for a given protease and characterize their cleavage kinetics. The utility of CLiPS was demonstrated by determining the substrate specificity of two unrelated proteases, caspase-3 and enteropeptidase (or enterokinase). CLiPS unambiguously identified the caspase-3 consensus cleavage sequence DXVDG. Enteropeptidase was unexpectedly promiscuous, but exhibited a preference for substrates with the motif (D/E)RM, which were cleaved substantially faster than the canonical DDDDK recognition sequence, widely used for protein purification. CLiPS provides a straightforward and versatile approach to determine protease specificity and discover optimal substrates on the basis of cleavage kinetics.     

Biophysical analysis of the structural evolution of substrate specificity in RuBisCO

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant enzyme on Earth. However, its catalytic rate per molecule of protein is extremely slow and the binding of the primary substrate, CO₂, is competitively displaced by O_2. Hence, carbon fixation by RuBisCO is highly inefficient; indeed, in higher C3 plants, about 30% of the time the enzyme mistakes CO₂ for O₂. Using genomic and structural analysis, we identify regions around the catalytic site that play key roles in discriminating between CO₂ and O₂. Our analysis identified positively charged cavities directly around the active site, which are expanded as the enzyme evolved with higher substrate specificity. The residues that extend these cavities have recently been under selective pressure, indicating that larger charged pockets are a feature of modern RuBisCOs, enabling greater specificity for CO₂. This paper identifies a key structural feature that enabled the enzyme to evolve improved CO₂ sequestration in an oxygen-rich atmosphere and may guide the engineering of more efficient RuBisCOs.

With an estimated mass of approximately 0.7 × 10¹⁵ g, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39) is almost certainly the most abundant enzyme on Earth (1). The enzyme catalyzes the addition of carbon dioxide (CO₂) from the environment to ribulose 1,5-bisphosphate (RuBP) to form two molecules of 3-phosphoglycerate (i.e., carboxylation), each of which subsequently are reduced to an aldehyde in the Calvin–Benson–Bassham cycle (2). In the contemporary world, the enzyme is, by far, the most important carbon fixing enzyme and, hence, the source of organic carbon for all animals and many microorganisms (3) on this planet. However, RuBisCO is an ancient enzyme; phylogenetic analyses suggest it evolved from a noncarbon fixing ancestral enzyme (4, 5) and is found in all domains of life (4, 6).Unlike many other carboxylating enzymes, RuBisCO only reacts with CO₂ rather than bicarbonate (HCO₃⁻). Curiously, the carboxylation reaction of RuBisCO is competitively inhibited by molecular oxygen (7). The two gases have very different structures. Nevertheless, the competitive binding between O₂ and CO₂ to RuBisCO severely hinders the efficiency of the enzyme in the contemporary atmosphere where CO₂ concentrations are relatively low and O₂ concentrations are relatively high (8, 9). How the enzyme “mistakes” O₂ for CO₂ is poorly understood.The discrimination between O₂ and CO₂ can be quantitatively described by the specificity ratio, S, which is defined as:S =VcKo/VoKc,where V_c and V_o are maximal velocities for carboxylation and oxygenation, respectively, and K_c and K_o are the relative Michaelis constants for CO₂ and O₂, respectively (2, 9). Due to the poor selectivity of RuBisCO and low turnover rate, the enzyme’s performance limits global carbon fixation (10, 11). Consequently, RuBisCO has become a major target for artificial engineering to enhance both catalytic rate and substrate specificity in higher plants to boost crop and water use efficiency (12).To date, four distinct groups of the enzyme have been identified (6, 13). All have a homodimeric functional form composed of two large (L) subunits (∼50–55 kDa) and some have attached small (S) subunits (∼10–12 kDa) (14). Depending on the type of RuBisCO, the holoenzyme could be anywhere from a simple homodimer (e.g., group II [L2] and group III [L2]) to a hexadecamer (the most widely distributed form) (e.g., group I [L8S8] and group III [L8]) and octadecamer (e.g., group III [L10]) (4, 6). The catalytic site of the enzyme resides in the large subunit in an α/β domain which contains Mg²⁺, which acts to stabilize and polarize the substrates during catalysis.The first step of catalysis is activation by carbamylating the catalytic lysine in the active site that binds to Mg²⁺ (14). Upon activation, the enzyme accepts its substrate, RuBP, to be enolized to a 2,3-enediolate intermediate, where a second CO₂ eventually binds (15). This six-carbon intermediate is further, sequentially hydrated, cleaved, and protonated to yield two molecules of phosphoglyceric acid (9, 14–16). The 2,3-enediolate is sensitive to the presence of O₂, which presents challenges to the enzyme’s bifunctional property to either take the carboxylation or oxygenation route.What controls this bifunctional property?The evolutionary history of RuBisCO can potentially reveal the key amino acids that led to increased catalytic selectivity (17). Indeed, the composition of the atmosphere has changed dramatically over the course of Earth’s history, in part due to RuBisCO activity (8, 17). RuBisCO evolved under anaerobic conditions (5, 6), in the Archean Eon, prior to the Great Oxidation Event around 2.33 billion years ago (18). From the Archean to the mid-Phanerozoic, atmospheric CO₂ levels dropped by four orders of magnitude, while O₂ levels rose by three orders of magnitude (17). This evolution, from an anaerobic to an aerobic environment, challenged RuBisCO’s ability to maintain net carbon fixation rates amid diminishing substrate and increasing inhibitor availability.Photosynthetic organisms adopted at least three main strategies to improve net photosynthetic carbon fixation in the face of exacerbating O₂/CO₂: 1) improve RuBisCO specificity, 2) increase the intracellular CO₂ concentration through the expression of a carbon concentrating mechanism (CCM), or 3) inhabit an ecological niche which maintains a low O₂/CO₂ (19). These three strategies, which are not mutually exclusive, led to a spectrum of specificity in the extant enzyme. Especially the CCM in algae and its analog of C4 photosynthesis in higher plants allowed the enzyme to be virtually saturated with inorganic carbon, which, in some cases, led to a relaxation in specificity (17, 20, 21).Here, we examine the correlations between the genetics, amino acid sequences, and the structures of available RuBisCO to better understand what features conferred selectivity between the two substrates. Our results provide a mechanistic interpretation of substrate selectivity and suggest how RuBisCO can be genetically improved.     

The human bile salt export pump: characterization of substrate specificity and identification of inhibitors

BACKGROUND & AIMS: The bile salt export pump (BSEP) is the major bile salt transporter in the liver canalicular membrane. Our aim was to determine the affinity of the human BSEP for bile salts and identify inhibitors. METHODS: Human BSEP was expressed in insect cells. Adenosine triphosphatase (ATPase) assays were performed, and bile salt transport studies were undertaken. RESULTS: The BSEP gene, ABCB11, was cloned and a recombinant baculovirus was generated. Infected insect cells expressed a 140-kilodalton protein that was absent in uninfected and in mock-infected cells. An ATPase assay showed BSEP to have a high basal ATPase activity. Transport assays were used to determine the Michaelis constant for taurocholate as 4.25 micromol/L, with a maximum velocity of 200 pmol x min(-1) x mg(-1) protein. Inhibition constant values for other bile salts were 11 micromol/L for glycocholate, 7 micromol/L for glycochenodeoxycholate, and 28 micromol/L for taurochenodeoxycholate. Cyclosporin A, rifampicin, and glibenclamide were proved to be competitive inhibitors of BSEP taurocholate transport, with inhibition constant values of 9.5 micromol/L, 31 micromol/L, and 27.5 micromol/L, respectively. Progesterone and tamoxifen did not inhibit BSEP. CONCLUSIONS: The human BSEP is a high-affinity bile salt transporter. The relative affinities for the major bile salts differ from those seen in rodents and reflect the different bile salt pools. BSEP is competitively inhibited by therapeutic drugs. This is a potentially significant mechanism for drug-induced cholestasis.     

Anti-Arrhenius cleavage of covalent bonds in bottlebrush macromolecules on substrate

Spontaneous degradation of bottlebrush macromolecules on aqueous substrates was monitored by atomic force microscopy. Scission of C─C covalent bonds in the brush backbone occurred due to steric repulsion between the adsorbed side chains, which generated bond tension on the order of several nano-Newtons. Unlike conventional chemical reactions, the rate of bond scission was shown to decrease with temperature. This apparent anti-Arrhenius behavior was caused by a decrease in the surface energy of the underlying substrate upon heating, which results in a corresponding decrease of bond tension in the adsorbed macromolecules. Even though the tension dropped minimally from 2.16 to 1.89 nN, this was sufficient to overpower the increase in the thermal energy (k_BT) in the Arrhenius equation. The rate constant of the bond-scission reaction was measured as a function of temperature and surface energy. Fitting the experimental data by a perturbed Morse potential V = V₀(1 - e^-βx)² - fx, we determined the depth and width of the potential to be V₀ = 141 ± 19 kJ/mol and β^-1 = 0.18 ± 0.03 Å, respectively. Whereas the V₀ value is in reasonable agreement with the activation energy E_a = 80–220 kJ/mol of mechanical and thermal degradation of organic polymers, it is significantly lower than the dissociation energy of a C─C bond D_e = 350 kJ/mol. Moreover, the force constant K_x = 2β²V₀ = 1.45 ± 0.36 kN/m of a strained bottlebrush along its backbone is markedly larger than the force constant of a C─C bond K_l = 0.44 kN/m, which is attributed to additional stiffness due to deformation of the side chains.     

Phosphorylation of a UDP-glucuronosyltransferase regulates substrate specificity

UDP-glucuronosyltransferase (UGT) isozymes catalyze detoxification of numerous chemical toxins present in our daily diet and environment by conjugation to glucuronic acid. The special properties and enzymatic mechanism(s) that enable endoplasmic reticulum-bound UGT isozymes to convert innumerable structurally diverse lipophiles to excretable glucuronides are unknown. Inhibition of cellular UGT1A7 and UGT1A10 activities and of [33P]orthophosphate incorporation into immunoprecipitable proteins after exposure to curcumin or calphostin-C indicated that the isozymes are phosphorylated. Furthermore, inhibition of UGT phosphorylation and activity by treatment with PKCepsilon-specific inhibitor peptide supported PKC involvement. Co-immunoprecipitation, colocalization by means of immunofluorescence, and cross-linking studies of PKCepsilon and UGT1A7His revealed that the proteins reside within 11.4 angstroms of each other. Moreover, mutation of three PKC sites in each UGT isozyme demonstrated that T73A/G and T202A/G caused null activity, whereas S432G-UGT1A7 caused a major shift of its pH-8.5 optimum to 6.4 with new substrate selections, including 17beta-estradiol. S432G-UGT1A10 exhibited a minor pH shift without substrate alterations. PKCepsilon involvement was confirmed by the demonstration that PKCepsilon overexpression enhanced activity of UGT1A7 but not of its S432 mutant and the conversion of 17beta-[14C]estradiol by S432G-UGT1A7 but not by UGT1A7. Consistent with these observations, treatment of UGT1A7-transfected cells with PKCepsilon-specific inhibitor peptide or general PKC inhibitors increased 17beta-estradiol catalysis between 5- and 11-fold, with parallel decreases in phosphoserine-432. Here, we report a mechanism involving PKC-mediated phosphorylation of UGT such that phosphoserine/threonine regulates substrate specificity in response to chemical exposures, which possibly confers survival benefit.     

Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors

The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a “DFG-out” covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket.Receptor tyrosine kinases (RTKs) serve as critical sensors of extracellular cues that activate a myriad of intracellular signaling pathways to regulate cell state. There are 58 receptor tyrosine kinases in the human genome, and many have been demonstrated to be constitutively activated through amplification or mutation in particular cancers. The signals emanating from these RTKs, such as epidermal growth factor receptor (EGFR), FGF receptor (FGFR), platelet-derived growth factor receptor (PDGFR), protein kinase Kit (KIT), and protein kinase c-Met (MET), have been pharmacologically proven to be essential to the survival of cancers expressing mutant forms of these proteins. However, rapid resistance to monotherapy with first-generation RTK inhibitors has been universally observed. Resistance typically arises from the emergence of cancer cells expressing mutant forms of RTKs that are impervious to the action of first-generation drugs or from the activation of by-pass signaling mechanisms. Resistance can be overcome by developing new inhibitors that target the mutant RTK directly or target bypass signaling mechanisms. Indeed this approach has been deployed successfully in the case of resistance to first-generation inhibitors of EGFR in nonsmall cell lung cancer (NSCLC) and of Abelson tyrosine-protein kinase (ABL) in chronic myelogenous leukemia (CML) (1–4).Human FGFRs are a family of four RTKs (FGFR1–4) which are sensors of a diverse family of 18 FGF ligands. FGFRs are key regulators of fibrogenesis, embryogenesis, angiogenesis, metabolism, and many other processes of proliferation and differentiation (5, 6). The fundamental importance of FGFR to development is well proven by gain-of-function mutations that result in dwarfism in model organisms and in humans (7–10). Deregulation of FGFR signaling through mutation, chromosomal translocation, and gene amplification or overexpression has been documented abundantly in numerous cancers (11). Activation of FGFR-dependent signaling pathways can stimulate tumor initiation, progression, and resistance to therapy. Translocation events implicating the FGFR1 gene and various fusions of FGFR1 are found in myeloproliferative syndromes (12); chromosomal translocations of FGFR1 or FGFR3 and the transforming acidic coiled-coil genes (TACC1 or TACC3) are oncogenic in glioblastoma multiforme, bladder cancer, head and neck cancer, and lung cancer (13–16); oncogenic mutations of FGFR2 and FGFR3 are observed in lung squamous cell carcinoma; FGFR2 N549K is observed in 25% of endometrial cancers; FGFR3 t(4;14) alterations are reported in 15–20% of multiple myeloma (17–19); FGFR4 Y367C mutation in the transmembrane domain drives constitutive activation and enhanced tumorigenic phenotypes in a breast carcinoma cell line (20–22); and K535 and E550 mutants are reported to activate FGFR4 in rhabdomyosarcoma (23). FGFR amplification is reported in various cancers (24, 25): FGFR1 is amplified in colorectal, lung, and renal cell cancers (26, 27); FGFR2 is amplified in gastric cancer and colorectal cancer (28, 29); FGFR3 is commonly amplified in bladder cancer and also is reported for cervical, oral, and hematological cancers (30–32); and FGFR4 is amplified in hepatocellular carcinoma, gastric cancer, pancreatic cancer, and ovarian cancer (33–37). FGFR also is involved in autocrine activation of STAT3 as a positive feedback in many drug-treated cancer cells which are driven by diverse oncogenes such as EGFR, ALK, MET, and KRAS (38).Currently known inhibitors of kinases can target a variety of conformational states and binding pockets and can be either reversible or covalent. Several potent and selective ATP-competitive, small-molecule FGFR inhibitors have been reported, with BGJ398 and AZD4547 being the clinically most advanced compounds (Fig. 1A) (39–42). We previously reported the first (to our knowledge) covalent FGFR irreversible inhibitor (FIIN-1), which targets a cysteine residue conserved in all four FGFR kinases and which inhibits the proliferation of Ba/F3 cells engineered to be dependent on FGFR1, FGFR2, or FGFR3 with EC₅₀s in the 10-nM range, a potency comparable to that exhibited by BGJ398 and AZD4547 (43). All FGFR kinases have a valine at the gatekeeper position, in contrast to ABL, EGFR, KIT, and PDGFR, which all possess a threonine gatekeeper in which resistance can be conferred by mutation of the threonine to a larger hydrophobic valine, isoleucine, or methionine residue in response to first-generation inhibitors of these kinases (44–46). The FGFR V561M mutation was reported to induce strong resistance to PD173074 and FIIN-1 (43, 47); later the gatekeeper mutant FGFR3 V555M emerged as a mechanism of resistance to AZ8010 in KMS-11 myeloma cells and also was demonstrated to confer resistance to other FGFR inhibitors, including PD173074 and AZD4547 (48). The FGFR2 V564I gatekeeper mutant was isolated as a resistant clone in a FGFR2 Ba/F3 screen of dovitinib and also was reported to confer resistance to the multitargeted drug ponatinib (19). In humans the FGFR4 V550L gatekeeper mutation was detected in 9% (4/43) of embryonal rhabdomyosarcoma tumors (49), and the FGFR4 V550M mutation was detected in 13% (2/15) of neuroendocrine breast carcinomas (50). To overcome gatekeeper mutations found in primary FGFR-driven cancers and those that likely will arise in FGFR inhibitor-treated tumors in the future, we developed next-generation covalent FGFR inhibitors. Here we describe the identification and characterization of the covalent FGFR inhibitors FIIN-2 and FIIN-3, which to our knowledge are the first FGFR inhibitors that are capable of potently inhibiting the gatekeeper mutants of FGFRs. We also demonstrate that FIIN-3 is capable of covalently inhibiting both FGFR and EGFR by using distinct binding modes to target different cysteine residues.Open in a separate windowFig. 1.(A) Chemical structures of clinical-stage FGFR inhibitors. (B) Evolution of FIIN-2 and FIIN-3 from FIIN-1. Structures of the reversible counterparts FRIN-2 and FRIN-3 are shown also.     

Structural insight into substrate specificity of phosphodiesterase 10

Phosphodiesterases (PDEs) hydrolyze the second messengers cAMP and cGMP. It remains unknown how individual PDE families selectively recognize cAMP and cGMP. This work reports structural studies on substrate specificity. The crystal structures of the catalytic domains of the D674A and D564N mutants of PDE10A2 in complex with cAMP and cGMP reveal that two substrates bind to the active site with the same syn configuration but different orientations and interactions. The products AMP and GMP bind PDE10A2 with the anti configuration and interact with both divalent metals, in contrast to no direct contact of the substrates. The structures suggest that the syn configurations of cAMP and cGMP are the genuine substrates for PDE10 and the specificity is achieved through the different interactions and conformations of the substrates. The PDE10A2 structures also show that the conformation of the invariant glutamine is locked by two hydrogen bonds and is unlikely to switch for substrate recognition. Sequence alignment shows a potential pocket, in which variation of amino acids across PDE families defines the size and shape of the pocket and thus determines the substrate specificity.     

The specificity of JAK3 kinase inhibitors

PF-956980 is a selective inhibitor of JAK3, related in structure to CP-690550, a compound being evaluated in clinical trials for rheumatoid arthritis and prevention of allograft rejection. PF-956980 has been evaluated against a panel of 30 kinases, and found to have nanomolar potency against only JAK3. Cellular and whole blood activity of this compound parallels its potency and selectivity in enzyme assays. It was effective in vivo at inhibiting the delayed type hypersensivity reaction in mice. We compared 2 commercially available JAK3 inhibitors (WHI-P131 and WHI-P154) in the same panel of biochemical and cellular assays and found them to be neither potent nor selective for JAK3. Both were found to be nanomolar inhibitors of the EGF receptor family of kinases. As these compounds have been used in numerous publications in the transplant and autoimmune disease literature, their specificity should be considered when interpreting these results.     

Metal-coordinating substrate analogs as inhibitors of metalloenzymes.

A group of active-site metal coordinating inhibitors of zinc proteases (carboxypeptidase A, thermolysin, Bacillus cereus neutral protease, and angiotensin-converting enzyme) have been synthesized and their properties investigated. Their general structures are R-SH and R-NH-PO2(O phi)H, where-S- or -O- serve as metal ligands and R refers to an amino acid or peptide group designed to interact with substrate recognition sites. These inhibitors can be extremely potent; thus, N-(2-mercaptoacetyl)-D-phenylalanine, e.g., inhibits carboxypeptidase A with a Kiapp of 2.2 x 10(-7) M. The spectral response of cobalt(II)-substituted thermolysin or carboxypeptidase A to the sulfur-containing inhibitors signals the direct interaction of the mercaptan with the metal. An S leads to Co(II) charge transfer band is generated near 340 nm and is detected by absorption, circular dichroism, and magnetic circular dichroism. The cobalt(II) spectra indicate both inner sphere coordination with sulfur and 4-coordination in the enzyme-inhibitor complex. Thus, the metal undergoes a simple substitution reaction, the inhibitor most likely displacing water at the fourth coordination site.     

Structural and functional basis of protein phosphatase 5 substrate specificity

The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone- and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP–substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper- or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.Protein phosphatase 5 (PP5) has pleiotropic roles in cellular signaling, including DNA damage repair, proliferation of breast cancer cells, circadian cycling, response to cytotoxic stresses, Rac-dependent potassium ion channel activity, and activation of steroid hormone receptors [e.g., glucocorticoid receptor (GR) and estrogen receptor] (1, 2). It is a member of the phosphoprotein phosphatase (PPP) family of serine/threonine phosphatases, which has members that share a highly conserved catalytic core and catalytic mechanism dependent on two metal ions, commonly Mn²⁺. Most PPP family members exhibit high, nonspecific phosphatase activity. Specificity is provided by a large cohort of regulatory and other interacting proteins that function to inhibit basal activity and recruit substrates, thereby finely tuning the enzymes (3). This combinatorial approach enables a small number of catalytic subunits to have the breadth of specificity equivalent to that seen in kinases, which are greater in number by an order of magnitude. Structures of complexes between regulatory and catalytic domains have illuminated the importance of regulatory subunits in facilitating substrate recruitment (3). However, to date, there is no structural information describing how a substrate binds at the active site of a PPP; therefore, a central question remains of how local interactions between the substrate and the catalytic domain contribute to the molecular basis of dephosphorylation.PP5 is unique among the PPP family because it has a low basal activity caused by an autoinhibitory N-terminal tetratricopeptide (TPR) domain (4). Its activity is promoted by a number of cellular factors, including fatty acids and the molecular chaperone heat shock protein 90 (Hsp90) (5), both of which release autoinhibition by interacting with the TPR domain (6, 7). Many established PP5 substrates are dependent on Hsp90 for their activation (known as Hsp90 clients). In addition to a requirement for Hsp90’s chaperone activity, it is likely that these PP5 substrates require Hsp90 to act as a molecular bridge to bring the catalytic domain of PP5 in close proximity to enable dephosphorylation, which has been shown for the Hsp90 cochaperone Cdc37 (8). In such cases, Hsp90 performs a role similar to that observed by the regulatory subunits of the PP1 and PP2A family (3).The cochaperone Cdc37 regulates the activation of Hsp90 client kinases by distinguishing between client and nonclient kinases (9) and recruiting the former to Hsp90 (10). Many of these kinases are oncogenes; therefore, the molecular details of their activation are of considerable interest in cancer therapy. Activation is dependent on a cycle of Cdc37-Ser13 phosphorylation by the constitutively active kinase CK2 (11, 12) and dephosphorylation by PP5 (8). The mechanisms by which Cdc37 phosphorylation and dephosphorylation regulate kinase activation are not understood.To understand the molecular determinants of the phospho-Ser13 Cdc37-PP5 interaction, we determined the 2.3-Å crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals how PP5 uses conserved elements of PPP structure to bind substrate, whereas in vitro and in vivo analyses indicate that, despite being highly conserved, elements of substrate specificity are determined within the phosphatase catalytic domain itself.     

Designing substrate specificity by protein engineering of electrostatic interactions.

Protein engineering of electrostatic interactions between charged substrates and complementary charged amino acids, at two different sites in the substrate binding cleft of the protease subtilisin BPN', increases kcat/Km toward complementary charged substrates (up to 1900 times) and decreases kcat/Km toward similarly charged substrates. From kinetic analysis of 16 mutants of subtilisin and the wild type, the average free energies for enzyme-substrate ion-pair interactions at the two different sites are calculated to be -1.8 +/- 0.5 and -2.3 +/- 0.6 kcal/mol (1 cal = 4.18 J) [at 25 degrees C in 0.1 M Tris X HCl (pH 8.6)]. The combined electrostatic effects are roughly additive. These studies demonstrate the feasibility for rational design of charged ligand binding sites in proteins by tailoring of electrostatic interactions.     

Target-induced formation of neuraminidase inhibitors from in vitro virtual combinatorial libraries

Neuraminidase, a key enzyme responsible for influenza virus propagation, has been used as a template for selective synthesis of small subsets of its own inhibitors from theoretically highly diverse dynamic combinatorial libraries. We show that the library building blocks, aldehydes and amines, form significant amounts of the library components resulting from their coupling by reductive amination only in the presence of the enzyme. The target amplifies the best hits at least 120-fold. The dynamic libraries synthesized and screened in such an in vitro virtual mode form the components that possess high inhibitory activity, as confirmed by enzyme assays with independently synthesized individual compounds.     

Global analysis of chaperone effects using a reconstituted cell-free translation system

Protein folding is often hampered by protein aggregation, which can be prevented by a variety of chaperones in the cell. A dataset that evaluates which chaperones are effective for aggregation-prone proteins would provide an invaluable resource not only for understanding the roles of chaperones, but also for broader applications in protein science and engineering. Therefore, we comprehensively evaluated the effects of the major Escherichia coli chaperones, trigger factor, DnaK/DnaJ/GrpE, and GroEL/GroES, on ～800 aggregation-prone cytosolic E. coli proteins, using a reconstituted chaperone-free translation system. Statistical analyses revealed the robustness and the intriguing properties of chaperones. The DnaK and GroEL systems drastically increased the solubilities of hundreds of proteins with weak biases, whereas trigger factor had only a marginal effect on solubility. The combined addition of the chaperones was effective for a subset of proteins that were not rescued by any single chaperone system, supporting the synergistic effect of these chaperones. The resource, which is accessible via a public database, can be used to investigate the properties of proteins of interest in terms of their solubilities and chaperone effects.     

Prediction and experimental validation of enzyme substrate specificity in protein structures

Structural Genomics aims to elucidate protein structures to identify their functions. Unfortunately, the variation of just a few residues can be enough to alter activity or binding specificity and limit the functional resolution of annotations based on sequence and structure; in enzymes, substrates are especially difficult to predict. Here, large-scale controls and direct experiments show that the local similarity of five or six residues selected because they are evolutionarily important and on the protein surface can suffice to identify an enzyme activity and substrate. A motif of five residues predicted that a previously uncharacterized Silicibacter sp. protein was a carboxylesterase for short fatty acyl chains, similar to hormone-sensitive-lipase–like proteins that share less than 20% sequence identity. Assays and directed mutations confirmed this activity and showed that the motif was essential for catalysis and substrate specificity. We conclude that evolutionary and structural information may be combined on a Structural Genomics scale to create motifs of mixed catalytic and noncatalytic residues that identify enzyme activity and substrate specificity.As the list of known genes grows exponentially, the elucidation of their function remains a major bottleneck and lags far behind the production of sequences (1–5). The best approach remains to search computationally for functionally characterized sequence homologs, ideally with greater than 50% sequence identity (6). Binding specificity, however, is sensitive to subtle amino acid differences, and the transfer of substrate between related enzymes is prone to errors when sequence identity is below 65–80% (7–9). These thresholds vary from case to case: Some orthologs will maintain identical functions down to 25% sequence identify (9), whereas paralogs can take on highly diverse activities (10). Other difficulties that plague annotation transfer between homologs are that individual small molecules may each bind to multiple and distinct molecular pockets (11), that different residues can support similar chemistries (12), and that activity can vary even when catalytic residues are conserved (13–18). To raise annotation accuracy, Structural Genomics (19) made structural information widely available and spurred the development of annotation methods dependent on local chemical and physical environments (20), sequence and structural comparisons (21), or 3D templates (22). In the case of the latter, these methods search between proteins for local structural similarities over a few signature residues that represent the telltale parts of a functional site, so-called “3D templates” (3, 14, 18, 22–24). The residue composition of 3D templates is critical, however, and derived from experiments (25) or from analyses of functional sites and determinants (14, 15, 26). The sensitivity and specificity of template-based annotations still needs to be established experimentally (27, 28), but retrospective controls suggest they often predict enzyme catalytic activity (14, 16, 17, 29, 30).Here, to extend the functional resolution of 3D template annotations to substrates, we exploit Evolutionary Tracing (ET) (31, 32). ET ranks sequence positions by the tendency of their evolutionary variations to correlate with major or with minor divergences. Top-ranked ET sequence positions are the most evolutionarily and, presumably, functionally important, and indeed they map out functional sites and specificity determinants (33) accurately enough to efficiently design mutations that block or swap functions among homologs in vitro (34–36) or in vivo (37, 38).Accordingly, given a query protein of unknown function, the ET Annotation pipeline (ETA) builds a 3D template from five or six top-ranked ET residues that also cluster together on surface regions of protein structures (31, 32). ETA then searches already annotated protein structures, the targets, for those that match the query 3D template (Fig. 1 and Movie S1). False positive matches are common but can be recognized because they typically (i) involve unimportant residues in the target (39), (ii) are not reciprocated back to the query (40), and (iii) point to multiple proteins that each bear unrelated functions. With appropriate specificity filters to eliminate these false positives, ETA identified enzyme activity down to the first three Enzyme Commission (EC) levels with 92% accuracy (40), as well as in nonenzymes (41) in large-scale Structural Genomics retrospective controls. The prediction of substrate specificity remains an open question and further requires accurate identification of the fourth and last EC level (42) presumably by adding a more discriminating use of 3D template residues than is sufficient to specify a general chemical process (43). Some sequence methods (29, 30) and other structure methods (14, 44) have aimed to predict all four EC levels, but to our knowledge they have not been directly tested on de novo predictions of substrate specificity.Open in a separate windowFig. 1.ETA accurately determines substrate specificity. (A) The ET algorithm is applied to a protein from Sulfolobus tokadaii strain 7 (green, PDB ID code 2eer, chain A) to identify evolutionarily important residues. A cluster of 10 or more important residues is identified and a Template Picker algorithm further selects five or six residues to act as a template that is used to probe a target library of proteins with known functions. Paired-distance matching algorithm identifies regions in protein structures in the target library that are similar to the template. Found matches are next passed to the SVM, which identifies significant matches based on geometric and evolutionary similarities. ETA repeats all these steps reciprocally, generating templates from target structures and searching for matches in the query protein. Following this protocol, ETA suggests four matches: alcohol dehydrogenase from Saccharomyces cerevisae (blue left, PDB ID code 2hcy), alcohol dehydrogenase from S. solfataricus (blue middle, PDB ID code 1r37), human class II alcohol dehydrogenase (blue right, PDB ID code 3cos), and NADP(H)-dependent cinnamyl alcohol dehydrogenase from S. cerevisae (red, PDB ID code 1piw) to the query protein. (B) The most seen function among matches, alcohol dehydrogenase activity (EC 1.1.1.1), is identified with high confidence with a confidence value of 1.125 as calculated in the box. (C) Comparison of PPV versus confidence score binned at <1, =1, and >1 for both six-residue templates (Left) and five-residue templates (Right) when considering only matches of <30% sequence identity. For more detail, see Fig. S1. (D) Comparison of PPV when predictions are made using ETA or the closest structural match (TM-align). Horizontal axis shows the maximum sequence identity of matches for proteins depicted in corresponding bars; the vertical axis is the PPV for each bin range.In this study, we improve the functional resolution of the ETA pipeline to identify relevant functional homology down to very low sequence identity and add substrate specificity to its large-scale predictions. We then experimentally validate the predictions and show that both catalytic and noncatalytic residues are essential for 3D templates to pinpoint activity and substrate specificity.     

Presentation assessment of minor histocompatibility antigens by predictive proteasomal cleavage analysis

Minor histocompatibility peptides (mHps) derived from polymorphic segments of endogenous proteins are thought to be targets for graft-versus-host and graft-versus-leukemia reactions after HLA-identical stem cell transplantation. A great majority of antigenic peptides is generated by fragmentation of proteins in the course of proteasomal processing. An algorithm was recently developed to predict cleavage sites during proteasomal processing. We tested the accuracy of the algorithm to predict mHps using 18 amino acid (AA) sequences of minor histocompatibility antigens (mHags) encoded by autosomal genes representing single nucleotide polymorphisms or by Y-chromosomal genes. The algorithm correctly predicted the C-termini of 11 of 13 experimentally confirmed mHps: 1) Correct prediction of C- and N-termini, e.g., for HA-1^H; 2) Correct prediction of C- and N-termini while anticipating intra-epitope cleavage sites, e.g., for SMCY-A*0201; 3) Correct prediction of C-termini and N-terminal extensions, e.g., for HA-8^R/V; and 4) Correct prediction of C-termini and N-terminal extensions while anticipating intra-epitope cleavage sites, e.g., for UTY-B8. Analysis of experimentally unconfirmed allelic counterparts of four autosomal mHags showed that AA substitutions either led to the insertion of an epitope-destroying cleavage site (e.g., in HA-1^R) or abolished the correct C-terminus (e.g., in HA-2^M). The proteasomal processing algorithm provides reliable data on the generation of mHps and forecasts their presence or absence. Combined with MHC class I ligand prediction, it can be a useful tool for the prediction of generation and presentation of new CTL epitopes derived from minor histocompatibility antigens.Abbreviations AA Amino acid - APC Antigen presenting cell - C Carboxy - CTL Cytotoxic T lymphocyte - EBV-BLCL Epstein-Barr virus transformed B lymphoblastoid cell line - ER Endoplasmic reticulum - HLA Human leukocyte antigen - mHags Minor histocompatibility antigens, AA sequences providing minor histocompatibility peptides - MHC Major histocompatibility complex - mHps Peptides derived from minor histocompatibility antigens - N Amino - TAP Transporter associated with antigen processing