Putative structural rearrangements associated with the interaction of macrocyclic inhibitors with norovirus 3CL protease

Human noroviruses are the primary cause of outbreaks of acute gastroenteritis worldwide. The problem is further compounded by the current lack of norovirus-specific antivirals or vaccines. Noroviruses have a single-stranded, positive sense 7 to 8 kb RNA genome which encodes a polyprotein precursor that is processed by a virus-encoded 3C-like cysteine protease (NV 3CLpro) to generate at least six mature nonstructural proteins. Processing of the polyprotein is essential for virus replication, consequently, NV 3CLpro has emerged as an attractive target for the discovery of norovirus therapeutics and prophylactics. We have recently described the structure-based design of macrocyclic transition state inhibitors of NV 3CLpro. In order to gain insight and understanding into the interaction of macrocyclic inhibitors with the enzyme, as well as probe the effect of ring size on pharmacological activity and cellular permeability, additional macrocyclic inhibitors were synthesized and high resolution cocrystal structures determined. The results of our studies tentatively suggest that the macrocyclic scaffold may hamper optimal binding to the active site by impeding concerted cross-talk between the S₂ and S₄ subsites.     

Structural characterization of the third scavenger receptor cysteine‐rich domain of murine neurotrypsin

Neurotrypsin (NT) is a multi‐domain serine protease of the nervous system with only one known substrate: the large proteoglycan Agrin. NT has seen to be involved in the maintenance/turnover of neuromuscular junctions and in processes of synaptic plasticity in the central nervous system. Roles which have been tied to its enzymatic activity, localized in the C‐terminal serine‐protease (SP) domain. However the purpose of NT's remaining 3–4 scavenger receptor cysteine‐rich (SRCR) domains is still unclear. We have determined the crystal structure of the third SRCR domain of murine NT (mmNT‐SRCR3), immediately preceding the SP domain and performed a comparative structural analysis using homologous SRCR structures. Our data and the elevated degree of structural conservation with homologous domains highlight possible functional roles for NT SRCRs. Computational and experimental analyses suggest the identification of a putative binding region for Ca²⁺ ions, known to regulate NT enzymatic activity. Furthermore, sequence and structure comparisons allow to single out regions of interest that, in future studies, might be implicated in Agrin recognition/binding or in interactions with as of yet undiscovered NT partners.     

Crystal structure of the N domain of Lon protease from Mycobacterium avium complex

Lon protease is evolutionarily conserved in prokaryotes and eukaryotic organelles. The primary function of Lon is to selectively degrade abnormal and certain regulatory proteins to maintain the homeostasis in vivo. Lon mainly consists of three functional domains and the N‐terminal domain is required for the substrate selection and recognition. However, the precise contribution of the N‐terminal domain remains elusive. Here, we determined the crystal structure of the N‐terminal 192‐residue construct of Lon protease from Mycobacterium avium complex at 2.4 å resolution,and measured NMR‐relaxation parameters of backbones. This structure consists of two subdomains, the β‐strand rich N‐terminal subdomain and the five‐helix bundle of C‐terminal subdomain, connected by a flexible linker,and is similar to the overall structure of the N domain of Escherichia coli Lon even though their sequence identity is only 26%. The obtained NMR‐relaxation parameters reveal two stabilized loops involved in the structural packing of the compact N domain and a turn structure formation. The performed homology comparison suggests that structural and sequence variations in the N domain may be closely related to the substrate selectivity of Lon variants. Our results provide the structure and dynamics characterization of a new Lon N domain, and will help to define the precise contribution of the Lon N‐terminal domain to the substrate recognition.     

Solution structure and dynamics of human ubiquitin conjugating enzyme Ube2g2

Ube2g2 is an E2 enzyme which functions as part of the endoplasmic reticulum‐associated degradation (ERAD) pathway responsible for identification and degradation of misfolded proteins in the endoplasmic reticulum. In tandem with a cognate E3 ligase, Ube2g2 assembles K48‐linked polyubiquitin chains and then transfers them to substrate, leading ultimately to proteasomal degradation of the polyubiquitin‐tagged substrate. We report here the solution structure and backbone dynamics of Ube2g2 solved by nuclear magnetic resonance spectroscopy. Although the solution structure agrees well with crystallographic structures for the E2 core, catalytically important loops (encompassing residues 95–107 and 130–135) flanking the active site cysteine are poorly defined. ¹⁵N spin relaxation and residual dipolar coupling analysis directly demonstrates that these two loops are highly dynamic in solution. These results suggest that Ube2g2 requires one or more of its protein partners, such as cognate E3, acceptor ubiquitin substrate or thiolester‐linked donor ubiquitin, to assume its catalytically relevant conformation. Within the NMR structural ensemble, interactions were observed between His94 and the highly mobile loop residues Asp98 and Asp99, supporting a possible role for His94 as a general base activated by the carboxylate side‐chains of Asp98 or Asp99. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Crystal structure of a [NiFe] hydrogenase maturation protease HybD from Thermococcus kodakarensis KOD1

A [NiFe] hydrogenase maturation protease HybD from Thermococcus kodakarensis KOD1 (TkHybD) is involved in the cleavage of the C‐terminal residues of [NiFe] hydrogenase large subunits by Ni recognition. Here, we report the crystal structure of TkHybD at 1.82 Å resolution to better understand this process. TkHybD exhibits an α/β/α sandwich fold with conserved residues responsible for the Ni recognition. Comparisons of TkHybD with homologous proteins also reveal that they share a common overall architecture, suggesting that they have similar catalytic functions. Our results including metal binding site prediction provide insight into the substrate recognition and catalysis mechanism of TkHybD. Proteins 2016; 84:1321–1327. © 2016 Wiley Periodicals, Inc.     

Motional timescale predictions by molecular dynamics simulations: Case study using proline and hydroxyproline sidechain dynamics

We propose a new approach for force field optimizations which aims at reproducing dynamics characteristics using biomolecular MD simulations, in addition to improved prediction of motionally averaged structural properties available from experiment. As the source of experimental data for dynamics fittings, we use ¹³C NMR spin‐lattice relaxation times T₁ of backbone and sidechain carbons, which allow to determine correlation times of both overall molecular and intramolecular motions. For structural fittings, we use motionally averaged experimental values of NMR J couplings. The proline residue and its derivative 4‐hydroxyproline with relatively simple cyclic structure and sidechain dynamics were chosen for the assessment of the new approach in this work. Initially, grid search and simplexed MD simulations identified large number of parameter sets which fit equally well experimental J couplings. Using the Arrhenius‐type relationship between the force constant and the correlation time, the available MD data for a series of parameter sets were analyzed to predict the value of the force constant that best reproduces experimental timescale of the sidechain dynamics. Verification of the new force‐field (termed as AMBER99SB‐ILDNP) against NMR J couplings and correlation times showed consistent and significant improvements compared to the original force field in reproducing both structural and dynamics properties. The results suggest that matching experimental timescales of motions together with motionally averaged characteristics is the valid approach for force field parameter optimization. Such a comprehensive approach is not restricted to cyclic residues and can be extended to other amino acid residues, as well as to the backbone. Proteins 2014; 82:195–215. © 2013 Wiley Periodicals, Inc.     

NMR investigations of structural and dynamics features of natively unstructured drug peptide – salmon calcitonin: implication to rational design of potent sCT analogs

Backbone dynamics and conformational properties of drug peptide salmon calcitonin have been studied in aqueous solution using nuclear magnetic resonance (NMR). Although salmon calcitonin (sCT) is largely unfolded in solution (as has been reported in several circular dichroism studies), the secondary H^α chemical shifts and three bond H^N–H^α coupling constants indicated that most of the residues of the peptide are populating the α‐helical region of the Ramachandran (?, ψ) map. Further, the peptide in solution has been found to exhibit multiple conformational states exchanging slowly on the NMR timescale (10²–10³ s^?1), inferred by the multiple chemical shift assignments in the region Leu4–Leu12 and around Pro23 (for residues Gln20–Tyr22 and Arg24). Possibly, these slowly exchanging multiple conformational states might inhibit symmetric self‐association of the peptide and, in part, may account for its reduced aggregation propensity compared with human calcitonin (which lacks this property). The ¹⁵N NMR‐relaxation data revealed (i) the presence of slow (microsecond‐to‐millisecond) timescale dynamics in the N‐terminal region (Cys1–Ser5) and core residues His17 and Asn26 and (ii) the presence of high frequency (nanosecond‐to‐picosecond) motions in the C‐terminal arm. Put together, the various results suggested that (i) the flexible C‐terminal of sCT (from Thr25–Thr31) is involved in identification of specific target receptors, (ii) whereas the N‐terminal of sCT (from Cys1–Gln20) in solution – exhibiting significant amount of conformational plasticity and strong bias towards biologically active α‐helical structure – facilitates favorable conformational adaptations while interacting with the intermembrane domains of these target receptors. Thus, we believe that the structural and dynamics features of sCT presented here will be useful guiding attributes for the rational design of biologically active sCT analogs. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Is there nascent structure in the intrinsically disordered region of troponin I?

In striated muscle, the binding of calcium to troponin C (TnC) results in the removal of the C‐terminal region of the inhibitory protein troponin I (TnI) from actin. While structural studies of the muscle system have been successful in determining the overall organization of most of the components involved in force generation at the atomic level, the structure and dynamics of the C‐terminal region of TnI remains controversial. This domain of TnI is highly flexible, and it has been proposed that this intrinsically disordered region (IDR) regulates contraction via a “fly‐casting” mechanism. Different structures have been presented for this region using different methodologies: a single α‐helix, a “mobile domain” containing a small β‐sheet, an unstructured region, and a two helix segment. To investigate whether this IDR has in fact any nascent structure, we have constructed a skeletal TnC‐TnI chimera that contains the N‐domain of TnC (1–90), a short linker (GGAGG), and the C‐terminal region of TnI (97–182) and have acquired ¹⁵N NMR relaxation data for this chimera. We compare the experimental relaxation parameters with those calculated from molecular dynamic simulations using four models based upon the structural studies. Our experimental results suggest that the C‐terminal region of TnI does not contain any defined secondary structure, supporting the “fly‐casting” mechanism. We interpret the presence of a “plateau” in the ¹⁵N NMR relaxation data as being an intrinsic property of IDRs. We also identified a more rigid adjacent region of TnI that has implications for muscle performance under ischemic conditions. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

A norovirus protease structure provides insights into active and substrate binding site integrity

Norovirus 3C-like proteases are crucial to proteolytic processing of norovirus polyproteins. We determined the crystal structure of the 3C-like protease from Chiba virus, a norovirus, at 2.8-A resolution. An active site including Cys139 and His30 is present, as is a hydrogen bond network that stabilizes the active site conformation. In the oxyanion hole backbone, a structural difference was observed probably upon substrate binding. A peptide substrate/enzyme model shows that several interactions between the two components are critical for substrate binding and that the S1 and S2 sites appropriately accommodate the substrate P1 and P2 residues, respectively. Knowledge of the structure and a previous mutagenesis study allow us to correlate proteolysis and structure.     

A structural study of norovirus 3C protease specificity: binding of a designed active site-directed peptide inhibitor

Noroviruses are the major cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.7 ? resolution. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, which is based on the most rapidly cleaved recognition sequence in the 200 kDa polyprotein substrate, reacts covalently through its propenyl ethyl ester group (X) with the active site nucleophile, Cys 139. The structure permits, for the first time, the identification of substrate recognition and binding groups in a noroviral 3C protease and thus provides important new information for the development of antiviral prophylactics.     

C‐terminal domains of bacterial proteases: structure,function and the biotechnological applications

C‐terminal domains widely exist in the C‐terminal region of multidomain proteases. As a β‐sandwich domain in multidomain protease, the C‐terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C‐terminal protease domains are described. Furthermore, the application prospects of C‐terminal domains, including polycystic kidney disease, prepeptidase C‐terminal and collagen‐binding domain, in the area of medicine and biological artificial materials are also discussed.     

Broad-Spectrum Antivirals against 3C or 3C-Like Proteases of Picornaviruses,Noroviruses, and Coronaviruses

Phylogenetic analysis has demonstrated that some positive-sense RNA viruses can be classified into the picornavirus-like supercluster, which includes picornaviruses, caliciviruses, and coronaviruses. These viruses possess 3C or 3C-like proteases (3Cpro or 3CLpro, respectively), which contain a typical chymotrypsin-like fold and a catalytic triad (or dyad) with a Cys residue as a nucleophile. The conserved key sites of 3Cpro or 3CLpro may serve as attractive targets for the design of broad-spectrum antivirals for multiple viruses in the supercluster. We previously reported the structure-based design and synthesis of potent protease inhibitors of Norwalk virus (NV), a member of the Caliciviridae family. We report herein the broad-spectrum antiviral activities of three compounds possessing a common dipeptidyl residue with different warheads, i.e., an aldehyde (GC373), a bisulfite adduct (GC376), and an α-ketoamide (GC375), against viruses that belong to the supercluster. All compounds were highly effective against the majority of tested viruses, with half-maximal inhibitory concentrations in the high nanomolar or low micromolar range in enzyme- and/or cell-based assays and with high therapeutic indices. We also report the high-resolution X-ray cocrystal structures of NV 3CLpro-, poliovirus 3Cpro-, and transmissible gastroenteritis virus 3CLpro- GC376 inhibitor complexes, which show the compound covalently bound to a nucleophilic Cys residue in the catalytic site of the corresponding protease. We conclude that these compounds have the potential to be developed as antiviral therapeutics aimed at a single virus or multiple viruses in the picornavirus-like supercluster by targeting 3Cpro or 3CLpro.     

Crystal structure of the human CNOT6L nuclease domain reveals strict poly(A) substrate specificity

CCR4, an evolutionarily conserved member of the CCR4–NOT complex, is the main cytoplasmic deadenylase. It contains a C‐terminal nuclease domain with homology to the endonuclease‐exonuclease‐phosphatase (EEP) family of enzymes. We have determined the high‐resolution three‐dimensional structure of the nuclease domain of CNOT6L, a human homologue of CCR4, by X‐ray crystallography using the single‐wavelength anomalous dispersion method. This first structure of a deadenylase belonging to the EEP family adopts a complete α/β sandwich fold typical of hydrolases with highly conserved active site residues similar to APE1. The active site of CNOT6L should recognize the RNA substrate through its negatively charged surface. In vitro deadenylase assays confirm the critical active site residues and show that the nuclease domain of CNOT6L exhibits full Mg²⁺‐dependent deadenylase activity with strict poly(A) RNA substrate specificity. To understand the structural basis for poly(A) RNA substrate binding, crystal structures of the CNOT6L nuclease domain have also been determined in complex with AMP and poly(A) DNA. The resulting structures suggest a molecular deadenylase mechanism involving a pentacovalent phosphate transition.     

Structure of the N‐terminal domain of the metalloprotease PrtV from Vibrio cholerae

The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre‐pro‐protein that undergoes several N‐ and C‐terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N‐terminal domain (residues 23–103) that contains two short α‐helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C‐terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.     

The crystal structure of methenyltetrahydromethanopterin cyclohydrolase from Methanobrevibacter ruminantium

Methenyltetrahydromethanopterin cyclohydrolase (Mch) is involved in the methanogenesis pathway of archaea as a C₁ unit carrier where N⁵‐formyl‐tetrahydromethanopterin is converted to methenyl‐tetrahydromethanopterin. Mch from Methanobrevibacter ruminantium was cloned, purified, crystallized and its crystal structure solved at 1.37 Å resolution. A biologically active trimer, the enzyme is composed of two domains including an N‐terminal domain of six α‐helices encompassing a series of four β‐sheets and a predominantly anti‐parallel β–sheet at the C‐terminus flanked on one side by α‐helices. Sequence and structural alignments have helped identify residues involved in substrate binding and trimer formation. Proteins 2013; 81:2064–2070. © 2013 Wiley Periodicals, Inc.     

C‐terminal tail derived from the neighboring subunit is critical for the activity of Thermoplasma acidophilumD‐aldohexose dehydrogenase

The D ‐aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a homotetrameric enzyme that catalyzes the oxidation of several D ‐aldohexoses, especially D ‐mannose. AldT comprises a unique C‐terminal tail motif (residues 247–255) that shuts the active‐site pocket of the neighboring subunit. The functional role of the C‐terminal tail of AldT has been investigated using mutational and crystallographic analyses. A total of four C‐terminal deletion mutants (Δ254, Δ253, Δ252, and Δ249) and two site‐specific mutants (Y86G and P254G) were expressed by Escherichia coli and purified. Enzymatic characterization of these mutants revealed that the C‐terminal tail is a requisite and that the interaction between Tyr86 and Pro254 is critical for enzyme activity. The crystal structure of the Δ249 mutant was also determined. The structure showed that the active‐site loops undergo a significant conformational change, which leads to the structural deformation of the substrate‐binding pocket. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Structure and DNA binding characteristics of the three‐Cys2His2 domain of mouse testis zinc finger protein

The C‐terminal three‐Cys₂His₂ zinc‐finger domain (TZD) of mouse testis zinc‐finger protein binds to the 5′‐TGTACAGTGT‐3′ at the Aie1 (aurora‐C) promoter with high specificity. Interestingly, the primary sequence of TZD is unique, possessing two distinct linkers, TGEKP and GAAP, and distinct residues at presumed DNA binding sites at each finger, especially finger 3. A K_d value of ～10^?8 M was obtained from surface plasmon resonance analysis for the TZD‐DNA complex. NMR structure of the free TZD showed that each zinc finger forms a typical ββα fold. On binding to DNA, chemical shift perturbations and the R₂ transverse relaxation rate in finger 3 are significantly smaller than those in fingers 1 and 2, which indicates that the DNA binding affinity in finger 3 is weaker. Furthermore, the shift perturbations between TZD in complex with the cognate DNA and its serial mutants revealed that both ADE7 and CYT8, underlined in 5′‐ATATGTACAGTGTTAT‐3′, are critical in specific binding, and the DNA binding in finger 3 is sequence independent. Remarkably, the shift perturbations in finger 3 on the linker mutation of TZD (GAAP mutated to TGEKP) were barely detected, which further indicates that finger 3 does not play a critical role in DNA sequence‐specific recognition. The complex model showed that residues important for DNA binding are mainly located on positions ?1, 2, 3, and 6 of α‐helices in fingers 1 and 2. The DNA sequence and nonsequence‐specific bindings occurring simultaneously in TZD provide valuable information for better understanding of protein–DNA recognition. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Crystal structure of Streptococcus pneumoniae Sp1610, a putative tRNA methyltransferase,in complex with S‐adenosyl‐L‐methionine

Streptococcus pneumoniae Sp1610, a Class‐I fold S‐adenosylmethionine (AdoMet)‐dependent methyltransferase, is a member of the COG2384 family in the Clusters of Orthologous Groups database, which catalyzes the methylation of N¹‐adenosine at position 22 of bacterial tRNA. We determined the crystal structure of Sp1610 in the ligand‐free and the AdoMet‐bound forms at resolutions of 2.0 and 3.0 Å, respectively. The protein is organized into two structural domains: the N‐terminal catalytic domain with a Class I AdoMet‐dependent methyltransferase fold, and the C‐terminal substrate recognition domain with a novel fold of four α‐helices. Observations of the electrostatic potential surface revealed that the concave surface located near the AdoMet binding pocket was predominantly positively charged, and thus this was predicted to be an RNA binding area. Based on the results of sequence alignment and structural analysis, the putative catalytic residues responsible for substrate recognition are also proposed.     

Structure–activity relationship of marinostatin,a serine protease inhibitor isolated from a marine organism

A 12‐residue MST isolated from a marine organism is a potent serine protease inhibitor that has a double cyclic structure composed of two ester linkages formed between the β‐hydroxyl and β‐carboxyl groups, Thr³‐Asp⁹ and Ser⁸‐Asp¹¹. MST was synthesized by a regioselective esterification procedure employing two sets of orthogonally removable side‐chain protecting groups for the Asp and Ser/Thr residues. In the MST molecule, there were no significant changes observed in yield by changing the order of esterification. SAR study of MST revealed that the minimum required structure for expressing the inhibitory activity is the sequence (1–9) in a monocyclic structure where Pro⁷ located in the ring plays a crucial role in keeping the structural rigidity. By applying the structural motif of MST, we rationally designed protease inhibitory specificities that differ from those of the natural product. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Detection of drug‐induced conformational change of a transmembrane protein in lipid bilayers using site‐directed spin labeling

As a target of antiviral drugs, the influenza A M2 protein has been the focus of numerous structural studies and has been extensively explored as a model ion channel. In this study, we capitalize on the expanding body of high‐resolution structural data available for the M2 protein to design and interpret site‐directed spin‐labeling electron paramagnetic resonance spectroscopy experiments on drug‐induced conformational changes of the M2 protein embedded in lipid bilayers. We obtained data in the presence of adamantane drugs for two different M2 constructs (M2TM 22–46 and M2TMC 23–60). M2TM peptides were spin labeled at the N‐terminal end of the transmembrane domain. M2TMC peptides were spin labeled site specifically at cysteine residues substituted for amino acids within the transmembrane domain (L36, I39, I42, and L43) and the C‐terminal amphipathic helix (L46, F47, F48, C50, I51, Y52, R53, F54, F55, and E56). Addition of adamantane drugs brought about significant changes in measured electron paramagnetic resonance spectroscopy environmental parameters consistent with narrowing of the transmembrane channel pore and closer packing of the C‐terminal amphipathic helices.