Defining residues involved in human rhinovirus 2A proteinase substrate recognition

The 2A proteinase (2A(pro)) of human rhinoviruses (HRVs) initiates proteolytic processing by cleaving between the C-terminus of VP1 and its own N-terminus. It subsequently cleaves the host protein eIF4GI. HRV2 and HRV14 2A(pro) cleave at IITTA *GPSD and DIKSY *GLGP on their respective polyproteins. The HRV2 2A(pro) cleavage site on eIF4GI is TLSTR *GPPR. We show that HRV2 2A(pro) can self-process at the eIF4GI cleavage sequence whereas HRV14 2A(pro) cannot, due to the presence of the arginine residue at P1. The mutations A104C or A104S in HRV14 2A(pro) restored cleavage when arginine was present at P1, although not to wild-type levels. These experiments define residues which determine substrate recognition in rhinoviral 2A(pro).     

Structure and dynamics of coxsackievirus B4 2A proteinase, an enyzme involved in the etiology of heart disease

The 2A proteinases (2A(pro)) from the picornavirus family are multifunctional cysteine proteinases that perform essential roles during viral replication, involving viral polyprotein self-processing and shutting down host cell protein synthesis through cleavage of the eukaryotic initiation factor 4G (eIF4G) proteins. Coxsackievirus B4 (CVB4) 2A(pro) also cleaves heart muscle dystrophin, leading to cytoskeletal dysfunction and the symptoms of human acquired dilated cardiomyopathy. We have determined the solution structure of CVB4 2A(pro) (extending in an N-terminal direction to include the C-terminal eight residues of CVB4 VP1, which completes the VP1-2A(pro) substrate region). In terms of overall fold, it is similar to the crystal structure of the mature human rhinovirus serotype 2 (HRV2) 2A(pro), but the relatively low level (40%) of sequence identity leads to a substantially different surface. We show that differences in the cI-to-eI2 loop between HRV2 and CVB4 2A(pro) translate to differences in the mechanism of eIF4GI recognition. Additionally, the nuclear magnetic resonance relaxation properties of CVB4 2A(pro), particularly of residues G1 to S7, F64 to S67, and P107 to G111, reveal that the substrate region is exchanging in and out of a conformation in which it occupies the active site with association and dissociation rates in the range of 100 to 1,000 s(-1). This exchange influences the conformation of the active site and points to a mechanism for how self-processing can occur efficiently while product inhibition is avoided.     

Characterization of a torovirus main proteinase

Viruses of the order Nidovirales encode huge replicase polyproteins. These are processed primarily by the chymotrypsin-like main proteinases (M(pro)s). So far, M(pro)s have been studied only for corona-, arteri-, and roniviruses. Here, we report the characterization of the M(pro) of toroviruses, the fourth main Nidovirus branch. Comparative sequence analysis of polyprotein 1a of equine torovirus (EToV) strain Berne, identified a serine proteinase domain, flanked by hydrophobic regions. Heterologous expression of this domain resulted in autoprocessing at flanking cleavage sites. N-terminal sequence analysis of cleavage products tentatively identified FxxQ downward arrow(S, A) as the substrate consensus sequence. EToV M(pro) combines several traits of its closest relatives. It has a predicted three-domain structure, with two catalytic beta-barrel domains and an additional C-terminal domain of unknown function. With respect to substrate specificity, the EToV M(pro) resembles its coronavirus homologue in its preference for P1-Gln, but its substrate-binding subsite, S1, more closely resembles that of arteri- and ronivirus M(pro)s, which prefer P1-Glu. Surprisingly, in contrast to the M(pro)s of corona- and roniviruses, but like that of arterivirus, the torovirus M(pro) uses serine instead of cysteine as its principal nucleophile. Under the premise that the M(pro)s of corona- and toroviruses are more closely related to each other than to those of arteri- and roniviruses, the transition from serine- to cysteine-based proteolytic catalysis (or vice versa) must have happened more than once in the course of nidovirus evolution. In this respect, it is of interest that a mutant EToV M(pro) with a Ser165-->Cys substitution retained partial enzymatic activity.     

Human rhinovirus 2 2Apro recognition of eukaryotic initiation factor 4GI. Involvement of an exosite

The 2A proteinase (2Apro) of human rhinovirus 2 is a cysteine proteinase with a unique chymotrypsin-like fold. During viral replication, 2Apro performs self-processing by cleaving between its own N terminus and the C terminus of the preceding protein, VP1. Subsequently, 2Apro cleaves the two isoforms of the cellular protein, eukaryotic initiation factor (eIF) 4G. We have previously shown that HRV2 2Apro can directly bind to eIF4G isoforms. Here we demonstrate using deletion mutants of eIF4GI that HRV2 2Apro requires eIF4GI amino acids 600-674 for binding; however, the amino acids at the cleavage site, Arg681 downward arrow Gly, are not required. The HRV2 2Apro binding domain for eIF4GI was identified by site-directed mutagenesis. Specifically, mutations Leu17 --> Arg and Asp35 --> Glu severely impaired HRV2 2Apro binding and thus processing of eIF4GI in rabbit reticulocyte lysates; self-processing, however, was not affected. Alanine scanning analysis further identified the loop containing residues Tyr32, Ser33, and Ser34 as important for eIF4GI binding. Although Asp35 is part of the catalytic triad, most of the eIF4GI binding domain lies in a unique exosite structure absent from other chymotrypsin-like enzymes and is distinct from the substrate binding cleft. The exosite represents a novel virulence determinant that may allow the development of specific inhibitors for HRV2 2Apro.     

Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes

The crystal structure of a pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. 101 (PSCP) has been solved by single-wavelength anomalous diffraction using the absorption peak of bromide anions. Structures of the uninhibited enzyme and of complexes with an inhibitor that was either covalently or noncovalently bound were refined at 1.0-1.4 A resolution. The structure of PSCP comprises a single compact domain with a diameter of approximately 55 A, consisting of a seven-stranded parallel beta-sheet flanked on both sides by a number of helices. The fold of PSCP is a superset of the subtilisin fold, and the covalently bound inhibitor is linked to the enzyme through a serine residue. Thus, the structure of PSCP defines a novel family of serine-carboxyl proteinases (defined as MEROPS S53) with a unique catalytic triad consisting of Glu 80, Asp 84 and Ser 287.     

The structure of yeast enolase at 2.25-A resolution. An 8-fold beta + alpha-barrel with a novel beta beta alpha alpha (beta alpha)6 topology

The three-dimensional structure of yeast enolase has been determined by the multiple isomorphous replacement method followed by the solvent flattening technique. A polypeptide model, corresponding with the known amino acid sequence, has been fitted to the electron density map. Crystallographic restrained least-squares refinement of the model without solvent gave R = 20.0% for 6-2.25-A resolution with good geometry. A model with 182 water molecules and 1 sulfate which is still being refined has presently R = 17.0%. The molecule is a dimer with subunits related by 2-fold crystallographic symmetry. The subunit has dimensions 60 X 55 X 45 A and is built from two domains. The smaller N-terminal domain has an alpha + beta structure based on a three-stranded antiparallel meander and four helices. The main domain is an 8-fold beta + alpha-barrel. The enolase barrel is, however, different from the triose phosphate isomerase barrel; its topology is beta beta alpha alpha (beta alpha)6 rather than (beta alpha)8 as found in triose phosphate isomerase. The inner beta-barrel is not entirely parallel, the second strand is antiparallel to the other strands, and the direction of the first helix is also reversed with respect to the other helices. This supports the hypothesis that some enzymes evolved independently producing the stable structure of beta alpha barrels with either enolase or triose phosphate isomerase topology. The active site of enolase is located at the carboxylic end of the barrel. A fragment of the N-terminal domain and two long loops protruding from the barrel domain form a wide crevice leading to the active site region. Asp246, Glu295, and Asp320 are the ligands of the conformational cation. Other residues in the active site region are Glu168, Asp321, Lys345, and Lys396.     

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.     

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding.     

Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain

The key enzyme in coronavirus polyprotein processing is the viral main proteinase, M(pro), a protein with extremely low sequence similarity to other viral and cellular proteinases. Here, the crystal structure of the 33.1 kDa transmissible gastroenteritis (corona)virus M(pro) is reported. The structure was refined to 1.96 A resolution and revealed three dimers in the asymmetric unit. The mutual arrangement of the protomers in each of the dimers suggests that M(pro) self-processing occurs in trans. The active site, comprised of Cys144 and His41, is part of a chymotrypsin-like fold that is connected by a 16 residue loop to an extra domain featuring a novel alpha-helical fold. Molecular modelling and mutagenesis data implicate the loop in substrate binding and elucidate S1 and S2 subsites suitable to accommodate the side chains of the P1 glutamine and P2 leucine residues of M(pro) substrates. Interactions involving the N-terminus and the alpha-helical domain stabilize the loop in the orientation required for trans-cleavage activity. The study illustrates that RNA viruses have evolved unprecedented variations of the classical chymotrypsin fold.     

Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: identification of a cis-replicating element in the coding sequence of 2A(pro)

We have previously shown that the RNA polymerase 3D(pol) of human rhinovirus 2 (HRV2) catalyzes the covalent linkage of UMP to the terminal protein (VPg) using poly(A) as a template (K. Gerber, E. Wimmer, and A. V. Paul, J. Virol. 75:10969-10978, 2001). The products of this in vitro reaction are VPgpU, VPgpUpU, and VPg-poly(U), the 5' end of minus-strand RNA. In the present study we used an assay system developed for poliovirus 3D(pol) (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74: 10359-10370, 2000) to search for a viral sequence or structure in HRV2 RNA that would provide specificity to this reaction. We now show that a small hairpin in HRV2 RNA [cre(2A)], located in the coding sequence of 2A(pro), serves as the primary template for HRV2 3D(pol) in the uridylylation of HRV2 VPg, yielding VPgpU and VPgpUpU. The in vitro reaction is strongly stimulated by the addition of purified HRV2 3CD(pro). Our analyses suggest that HRV2 3D(pol) uses a "slide-back" mechanism during synthesis of the VPg-linked precursors. The corresponding cis- replicating RNA elements in the 2C(ATPase) coding region of poliovirus type 1 Mahoney (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000) and VP1 of HRV14 (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) can be functionally exchanged in the assay with cre(2A) of HRV2. Mutations of either the first or the second A in the conserved A(1)A(2)A(3)CA sequence in the loop of HRV2 cre(2A) abolished both viral growth and the RNA's ability to serve as a template in the in vitro VPg uridylylation reaction.     

The structure of aconitase

The crystal structure of the 80,000 Da Fe-S enzyme aconitase has been solved and refined at 2.1 A resolution. The protein contains four domains; the first three from the N-terminus are closely associated around the [3Fe-4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Association of the larger C-terminal domain with the first three domains creates an extensive cleft leading to the Fe-S cluster. Residues from all four domains contribute to the active site region, which is defined by the Fe-S cluster and a bound SO4(2-) ion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well as several bound water molecules. Three of these side chains reside on a three-turn 3(10) helix in the first domain. The SO4(2-) ion is bound 9.3 A from the center of the [3Fe-4S] cluster by the side chains of 2 Arg and 1 Gln residues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.     

Three-dimensional structure of a barley beta-D-glucan exohydrolase, a family 3 glycosyl hydrolase.

BACKGROUND: Cell walls of the starchy endosperm and young vegetative tissues of barley (Hordeum vulgare) contain high levels of (1-->3,1-->4)-beta-D-glucans. The (1-->3,1-->4)-beta-D-glucans are hydrolysed during wall degradation in germinated grain and during wall loosening in elongating coleoptiles. These key processes of plant development are mediated by several polysaccharide endohydrolases and exohydrolases. RESULTS:. The three-dimensional structure of barley beta-D-glucan exohydrolase isoenzyme ExoI has been determined by X-ray crystallography. This is the first reported structure of a family 3 glycosyl hydrolase. The enzyme is a two-domain, globular protein of 605 amino acid residues and is N-glycosylated at three sites. The first 357 residues constitute an (alpha/beta)8 TIM-barrel domain. The second domain consists of residues 374-559 arranged in a six-stranded beta sandwich, which contains a beta sheet of five parallel beta strands and one antiparallel beta strand, with three alpha helices on either side of the sheet. A glucose moiety is observed in a pocket at the interface of the two domains, where Asp285 and Glu491 are believed to be involved in catalysis. CONCLUSIONS: The pocket at the interface of the two domains is probably the active site of the enzyme. Because amino acid residues that line this active-site pocket arise from both domains, activity could be regulated through the spatial disposition of the domains. Furthermore, there are sites on the second domain that may bind carbohydrate, as suggested by previously published kinetic data indicating that, in addition to the catalytic site, the enzyme has a second binding site specific for (1-->3, 1-->4)-beta-D-glucans.     

Alkaline proteinase inhibitor of Pseudomonas aeruginosa: a mutational and molecular dynamics study of the role of N-terminal residues in the inhibition of Pseudomonas alkaline proteinase

Alkaline proteinase inhibitor of Pseudomonas aeruginosa is a 11.5-kDa, high affinity inhibitor of the serralysin class of zinc-dependent proteinases secreted by several Gram-negative bacteria. X-ray crystallography of the proteinase-inhibitor complex reveals that five N-terminal inhibitor residues occupy the extended substrate binding site of the enzyme and that the catalytic zinc is chelated by the alpha-amino and carbonyl groups of the N-terminal residue of the inhibitor. In this study, we assessed the effect of alteration of inhibitor residues 2-5 on its affinity for Pseudomonas alkaline proteinase (APR) as derived from the ratio of the dissociation and associate rate constants for formation of the enzyme-inhibitor complex. The largest effect was observed at position Ser-2, which occupies the S1' pocket of the enzyme and donates a hydrogen bond to the carboxyl group of the catalytic Glu-177 of the proteinase. Substitution of Asp, Arg, or Trp at this position increased the dissociation constant KD by 35-, 180-, and 13-fold, respectively. Mutation at positions 3-5 of the trunk also resulted in a reduction in enzyme-inhibitor affinity, with the exception of an I4W mutant, which exhibited a 3-fold increase in affinity. Molecular dynamics simulation of the complex formation between the catalytic domain of APR and the S2D mutant showed that the carboxyl of Asp-2 interacts with the catalytic zinc, thereby partially neutralizing the negative charge that otherwise would clash with the carboxyl group of Glu-177 of APR. Simulation of the interaction between the alkaline proteinase and the I4W mutant revealed a major shift in the loop comprised of residues 189-200 of the enzyme that allowed formation of a stacking interaction between the aromatic rings of Ile-4 of the inhibitor and Tyr-158 of the proteinase. This new interaction could account for the observed increase in enzyme-inhibitor affinity.     

Analysis of picornavirus 2A(pro) proteins: separation of proteinase from translation and replication functions.

The poliovirus (PV) genome was manipulated by replacing its 2A-encoding sequence with the corresponding sequence of coxsackie B4 virus (CBV4) or human rhinovirus type 2 (HRV2). In vitro translation of the resulting chimeric PV genomes revealed a normal cis-cleavage activity for both heterologous 2A(pro) proteinases in the chimeric PV polyproteins. However, only the genome containing the 2A-encoding sequence of CBV4 (PV/CBV4-2A) yielded viable virus in transfected cells, producing a mixture of large and small plaques on HeLa cell monolayers. The large-plaque variants were found to contain single-amino-acid mutations at a specific site near the C terminus of the CBV4 2A(pro) protein. When the same single-amino-acid mutations were directly introduced into the parental PV/CBV4-2A genome, chimeric viruses with a large-plaque phenotype and a wild-type PV-like growth pattern were obtained upon transfection, an observation demonstrating that these point mutations alone had a drastic effect on the growth of the PV/CBV4 chimeric virus. On the other hand, the chimeric genome containing the 2A-encoding sequence of HRV2 (PV/HRV2-2A) produced a null phenotype in transfected HeLa cells, although low-level replication of this chimeric genome was evident. We conclude that only 2A(pro) of the more closely related enterovirus CBV4 is able to functionally substitute for that of PV in vivo, and a subtle genetic modification of the CBV4 2A(pro) protein results in a drastic improvement in the growth of the chimeric PV/CBV4-2A virus. In addition, this chimeric cDNA approach enabled us to dissect multiple biological functions encoded by the 2A(pro) proteins.     

Structure of human pro-chymase: a model for the activating transition of granule-associated proteases

Human chymase is a protease involved in physiological processes ranging from inflammation to hypertension. As are all proteases of the trypsin fold, chymase is synthesized as an inactive "zymogen" with an N-terminal pro region that prevents the transition of the zymogen to an activated conformation. The 1.8 A structure of pro-chymase, reported here, is the first zymogen with a dipeptide pro region (glycine-glutamate) to be characterized at atomic resolution. Three segments of the pro-chymase structure differ from that of the activated enzyme: the N-terminus (Gly14-Gly19), the autolysis loop (Gly142-Thr154), and the 180s loop (Pro185A-Asp194). The four N-terminal residues (Gly14-Glu15-Ile16-Ile17) are disordered. The autolysis loop occupies a position up to 10 A closer to the active site than is seen in the activated enzyme, thereby forming a hydrogen bond with the catalytic residue Ser195 and occluding the S1' binding pocket. Nevertheless, the catalytic triad (Asp102-His57-Ser195) is arrayed in a geometry close to that seen in activated chymase (all atom rmsd of 0.52 A). The 180s loop of pro-chymase is, on average, 4 A removed from its conformation in the activated enzyme. This conformation disconnects the oxyanion hole (the amides of Gly193 and Ser195) from the active site and positions only approximately 35% of the S1-S3 binding pockets in the active conformation. The backbone of residue Asp194 is rotated 180 degrees when compared to its conformation in the activated enzyme, allowing a hydrogen bond between the main-chain amide of residue Trp141 and the carboxylate of Asp194. The side chains of residues Phe191 and Lys192 of pro-chymase fill the Ile16 binding pocket and the base of the S1 binding pocket, respectively. The zymogen positioning of both the 180s and autolysis loops are synergistic structural elements that appear to prevent premature proteolysis by chymase and, quite possibly, by other dipeptide zymogens.     

Crystal structure of a deletion mutant of a tyrosyl-tRNA synthetase complexed with tyrosine

The crystal structure of a deletion mutant of tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been determined at 2.5 A resolution using molecular replacement techniques. The genetically engineered molecule catalyses the activation of tyrosine with kinetic properties similar to those of the wild-type enzyme but no longer binds tRNATyr. It contains 319 residues corresponding to the region of the polypeptide chain for which interpretable electron density is present in crystals of the wild-type enzyme. The partly refined model of the wild-type enzyme was used as a starting point in determining the structure of the truncated mutant. The new crystals are of space group P2(1) and contain the molecular dimer within the asymmetric unit. The refined model has a crystallographic R-factor of 18.7% for all reflections between 8 and 2.5 A. Each subunit contains two structural domains: the alpha/beta domain (residues 1 to 220) containing a six-stranded beta-sheet and the alpha-helical domain (residues 248 to 319) containing five helices. The alpha/beta domains are related by a non-crystallographic dyad while the alpha-helical domains are in slightly different orientations in the two subunits. The tyrosine substrate binds in a slot at the bottom of a deep active site cleft in the middle of the alpha/beta domain. It is surrounded by polar side-chains and water molecules that are involved in an intricate hydrogen bonding network. Both the alpha-amino and hydroxyl groups of the substrate make good hydrogen bonds with the protein. The amino group forms hydrogen bonds with Tyr169-OH, Asp78-OD1 and Gln173-OE1. The phenolic hydroxyl group forms hydrogen bonds with Asp76-OD1 and Tyr34-OH. In contrast, the substrate carboxyl group makes no direct interactions with the enzyme. The results of both substrate inhibition studies and site-directed mutagenesis experiments have been examined in the light of the refined structure.     

Cleavage of a bacterial autotransporter by an evolutionarily convergent autocatalytic mechanism

Bacterial autotransporters are comprised of an N-terminal 'passenger domain' and a C-terminal beta barrel ('beta domain') that facilitates transport of the passenger domain across the outer membrane. Following translocation, the passenger domains of some autotransporters are cleaved by an unknown mechanism. Here we show that the passenger domain of the Escherichia coli O157:H7 autotransporter EspP is released in a novel autoproteolytic reaction. After purification, the uncleaved EspP precursor underwent proteolytic processing in vitro. An analysis of protein topology together with mutational studies strongly suggested that the reaction occurs inside the beta barrel and revealed that two conserved residues, an aspartate within the beta domain (Asp(1120)) and an asparagine (Asn(1023)) at the P1 position of the cleavage junction, are essential for passenger domain cleavage. Interestingly, these residues were also essential for the proteolytic processing of two distantly related autotransporters. The data strongly suggest that Asp(1120) and Asn(1023) form an unusual catalytic dyad that mediates self-cleavage through the cyclization of the asparagine. Remarkably, a very similar mechanism has been proposed for the maturation of eukaryotic viral capsids.     

The nature of the catalytic domain of 2'-5'-oligoadenylate synthetases.

2'-5'-Oligoadenylate (2-5(A)) synthetases are a family of interferon-induced enzymes that are activated by double-stranded RNA. To understand why, unlike other DNA and RNA polymerases, they catalyze 2'-5' instead of 3'-5' phosphodiester bond formation, we used molecular modeling to compare the structure of the catalytic domain of DNA polymerase beta (pol beta) to that of a region of the P69 isozyme of 2-5(A) synthetase. Although the primary sequence identity is low, like pol beta, P69 can assume an alphabetabetaalphabetabetabeta structure in this region. Moreover, mutation of the three Asp residues of P69, which correspond to the three catalytic site Asp residues of pol beta, inactivated the enzyme without affecting its substrate and activator binding capacity, providing further credence to the concept that this region is the catalytic domain of P69. This domain is highly conserved among all 2-5(A) synthetase isozymes. Biochemical and mutational studies demonstrated that dimerization of the P69 protein is required for its enzyme activity. However, a dimer containing a wild type subunit and an inactive catalytic domain mutant subunit was also active. The rate of catalysis of the heterodimer was half of that of the wild type homodimer, although the two proteins bound double-stranded RNA and ATP equally well.     

Distinct structural and functional roles of conserved residues in the first extracellular domain of receptors for corticotropin-releasing factor and related G-protein-coupled receptors

The G-protein-coupled receptor B1 family includes corticotropin-releasing factor (CRF), growth hormone-releasing hormone, incretin, and pituitary adenylate cyclase-activating polypeptide receptors. The three-dimensional NMR structure of the first extracellular domain (ECD1) of CRF receptor 2beta (CRF-R2beta), free and complexed with astressin, comprises a Sushi domain. This domain is stabilized in part by a salt bridge between Asp(65) and Arg(101). Analogous residues are conserved in other members of the B1 family. To address the importance of the salt bridge residues within this receptor family, we studied the effects of mutating the residues in full-length CRF-R2beta and isolated ECD1. Mutation D65A or D65R/R101D resulted in loss of the canonical disulfide arrangement, whereas R101A retained the Cys(4)-Cys(6) disulfide bond. The mutations resulted in misfolding within the ECD1 as determined by NMR and 1-anilino-8-naphthalenesulfonate binding but did not prevent cell surface expression. The D65A mutation in CRF-R2beta greatly reduced binding and activation, but the R101A substitution had only a small effect. Similar effects were seen on astressin binding to the ECD1. The different interactions of Asp(65) and Arg(101), deduced from the three-dimensional structure of the complex, are consistent with the differential effects seen in the mutants. The reduction in binding of Asp(65) mutants is a consequence of a distinct Asp(65)-Trp(71) interaction, which stabilizes the ligand-binding loop. Hence, loss of the salt bridge leads to disruption of the overall fold but does not abolish function. Because homologous mutations in other B1 receptors produce similar effects, these conserved residues may play similar roles in the entire receptor family.     

Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinase with a caspase-like fold.

Gingipains are cysteine proteinases acting as key virulence factors of the bacterium Porphyromonas gingivalis, the major pathogen in periodontal disease. The 1.5 and 2.0 A crystal structures of free and D-Phe-Phe-Arg-chloromethylketone-inhibited gingipain R reveal a 435-residue, single-polypeptide chain organized into a catalytic and an immunoglobulin-like domain. The catalytic domain is subdivided into two subdomains comprising four- and six-stranded beta-sheets sandwiched by alpha-helices. Each subdomain bears topological similarities to the p20-p10 heterodimer of caspase-1. The second subdomain harbours the Cys-His catalytic diad and a nearby Glu arranged around the S1 specificity pocket, which carries an Asp residue to enforce preference for Arg-P1 residues. This gingipain R structure is an excellent template for the rational design of drugs with a potential to cure and prevent periodontitis. Here we show the binding mode of an arginine-containing inhibitor in the active-site, thus identifying major interaction sites defining a suitable pharmacophor.