Leader proteinase of beet yellows virus functions in long-distance transport

The 66-kDa leader proteinase (L-Pro) of the Beet yellows virus (BYV) possesses a nonconserved N-terminal domain and a conserved, papain-like C-terminal domain. Previous work revealed that the N-terminal domain functions in RNA amplification, whereas the C-terminal domain is required for autoproteolysis. Alanine-scanning mutagenesis was applied to complete the functional analysis of L-Pro throughout the virus life cycle. This analysis indicated that the C-terminal domain of L-Pro, in addition to being required for proteolysis, also functions in RNA amplification and that these two functions are genetically separable. Examination of the role of L-Pro in BYV cell-to-cell movement revealed that none of the 20 examined replication-competent mutants was movement defective. In contrast, six of the L-Pro mutations affected the long-distance transport of BYV to various degrees, whereas three mutations completely abolished the transport. Because these mutations were located throughout the protein molecule, both domains of L-Pro function in virus transport. We conclude that in addition to previously identified functions of L-Pro, it also serves as the BYV long-distance transport factor.     

Functional Specialization and Evolution of Leader Proteinases in the Family Closteroviridae

Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.     

The two-component NS2B-NS3 proteinase represses DNA unwinding activity of the West Nile virus NS3 helicase

Similar to many flavivirus types including Dengue and yellow fever viruses, the nonstructural NS3 multifunctional protein of West Nile virus (WNV) with an N-terminal serine proteinase domain and an RNA triphosphatase, an NTPase domain, and an RNA helicase in the C-terminal domain is implicated in both polyprotein processing and RNA replication and is therefore a promising drug target. To exhibit its proteolytic activity, NS3 proteinase requires the presence of the cofactor encoded by the upstream NS2B sequence. During our detailed investigation of the biology of the WNV helicase, we characterized the ATPase and RNA/DNA unwinding activities of the full-length NS2B-NS3 proteinase-helicase protein as well as the individual NS3 helicase domain lacking both the NS2B cofactor and the NS3 proteinase sequence and the individual NS3 proteinase-helicase lacking only the NS2B cofactor. We determined that both the NS3 helicase and NS3 proteinase-helicase constructs are capable of unwinding both the DNA and the RNA templates. In contrast, the full-length NS2B-NS3 proteinase-helicase unwinds only the RNA templates, whereas its DNA unwinding activity is severely repressed. Our data suggest that the productive, catalytically competent fold of the NS2B-NS3 proteinase moiety represents an essential component of the RNA-DNA substrate selectivity mechanism in WNV and, possibly, in other flaviviruses. Based on our data, we hypothesize that the mechanism we have identified plays a role yet to be determined in WNV replication occurring both within the virus-induced membrane-bound replication complexes in the host cytoplasm and in the nuclei of infected cells.     

The retroviral proteinase active site and the N-terminus of Ddi1 are required for repression of protein secretion

The Ddi1 protein of the yeast Saccharomyces cerevisiae is involved in numerous interactions with the ubiquitin system, which may be mediated by its N-terminal ubiquitin like domain and its C-terminal ubiquitin associated domain. Ddi1 also contains a central region with all the features of a retroviral aspartic proteinase, which was shown to be important in cell-cycle control. Here we demonstrate an additional role for this domain, along with the N-terminal region, in protein secretion. These results further substantiate the hypothesis that Ddi1 functions in vivo as a catalytically-active aspartic proteinase.     

A Compact Viral Processing Proteinase/Ubiquitin Hydrolase from the OTU Family

Turnip yellow mosaic virus (TYMV) - a member of the alphavirus-like supergroup of viruses - serves as a model system for positive-stranded RNA virus membrane-bound replication. TYMV encodes a precursor replication polyprotein that is processed by the endoproteolytic activity of its internal cysteine proteinase domain (PRO). We recently reported that PRO is actually a multifunctional enzyme with a specific ubiquitin hydrolase (DUB) activity that contributes to viral infectivity. Here, we report the crystal structure of the 150-residue PRO. Strikingly, PRO displays no homology to other processing proteinases from positive-stranded RNA viruses, including that of alphaviruses. Instead, the closest structural homologs of PRO are DUBs from the Ovarian tumor (OTU) family. In the crystal, one molecule''s C-terminus inserts into the catalytic cleft of the next, providing a view of the N-terminal product complex in replication polyprotein processing. This allows us to locate the specificity determinants of PRO for its proteinase substrates. In addition to the catalytic cleft, at the exit of which the active site is unusually pared down and solvent-exposed, a key element in molecular recognition by PRO is a lobe N-terminal to the catalytic domain. Docking models and the activities of PRO and PRO mutants in a deubiquitylating assay suggest that this N-terminal lobe is also likely involved in PRO''s DUB function. Our data thus establish that DUBs can evolve to specifically hydrolyze both iso- and endopeptide bonds with different sequences. This is achieved by the use of multiple specificity determinants, as recognition of substrate patches distant from the cleavage sites allows a relaxed specificity of PRO at the sites themselves. Our results thus shed light on how such a compact protein achieves a diversity of key functions in viral genome replication and host-pathogen interaction.     

Site-Specific Cleavage of the Host Poly(A) Binding Protein by the Encephalomyocarditis Virus 3C Proteinase Stimulates Viral Replication

Although picornavirus RNA genomes contain a 3'-terminal poly(A) tract that is critical for their replication, the impact of encephalomyocarditis virus (EMCV) infection on the host poly(A)-binding protein (PABP) remains unknown. Here, we establish that EMCV infection stimulates site-specific PABP proteolysis, resulting in accumulation of a 45-kDa N-terminal PABP fragment in virus-infected cells. Expression of a functional EMCV 3C proteinase was necessary and sufficient to stimulate PABP cleavage in uninfected cells, and bacterially expressed 3C cleaved recombinant PABP in vitro in the absence of any virus-encoded or eukaryotic cellular cofactors. N-terminal sequencing of the resulting C-terminal PABP fragment identified a 3C(pro) cleavage site on PABP between amino acids Q437 and G438, severing the C-terminal protein-interacting domain from the N-terminal RNA binding fragment. Single amino acid substitution mutants with changes at Q437 were resistant to 3C(pro) cleavage in vitro and in vivo, validating that this is the sole detectable PABP cleavage site. Finally, while ongoing protein synthesis was not detectably altered in EMCV-infected cells expressing a cleavage-resistant PABP variant, viral RNA synthesis and infectious virus production were both reduced. Together, these results establish that the EMCV 3C proteinase mediates site-specific PABP cleavage and demonstrate that PABP cleavage by 3C regulates EMCV replication.     

Structure-function relationships in the collagenase family member transin

We have developed a system for studying the proteinase activity of a collagenase family member, transin. Cos cells transfected with a vector designed to direct synthesis of a secretable fusion protein between staphylococcal protein A and transin secrete a latent proteinase, activable by 4-aminophenylmercuric acetate, which binds to IgG-Sepharose. Treatment with 4-aminophenylmercuric acetate leads to cleavage of the fusion protein and elution of the active proteinase transin. Based on results obtained with this system we propose that transin comprises an N-terminal proteinase domain and an independent C-terminal hemopexin-like domain. The latter domain is not required for binding of inhibitors or for maintenance of transin in its inactive form. The sequence PRCGVPDV is present in the proenzyme forms of collagenase family proteinases just upstream from the N termini of the active enzymes. We show that mutations within this sequence lead to transin variants with a much increased tendency to undergo spontaneous activation. Finally, we show that mutations within a region of transin having sequence similarity to the zinc-binding site of bacterial metalloproteinases inactivate the proteinase activity of transin, lending support to the notion that this region represents part of transin's active site.     

Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics.

The hepatitis C virus H strain (HCV-H) polyprotein is cleaved to produce at least 10 distinct products, in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. An HCV-encoded serine proteinase activity in NS3 is required for cleavage at four sites in the nonstructural region (3/4A, 4A/4B, 4B/5A, and 5A/5B). In this report, the HCV-H serine proteinase domain (the N-terminal 181 residues of NS3) was tested for its ability to mediate trans-processing at these four sites. By using an NS3-5B substrate with an inactivated serine proteinase domain, trans-cleavage was observed at all sites except for the 3/4A site. Deletion of the inactive proteinase domain led to efficient trans-processing at the 3/4A site. Smaller NS4A-4B and NS5A-5B substrates were processed efficiently in trans; however, cleavage of an NS4B-5A substrate occurred only when the serine proteinase domain was coexpressed with NS4A. Only the N-terminal 35 amino acids of NS4A were required for this activity. Thus, while NS4A appears to be absolutely required for trans-cleavage at the 4B/5A site, it is not an essential cofactor for serine proteinase activity. To begin to examine the conservation (or divergence) of serine proteinase-substrate interactions during HCV evolution, we demonstrated that similar trans-processing occurred when the proteinase domains and substrates were derived from two different HCV subtypes. These results are encouraging for the development of broadly effective HCV serine proteinase inhibitors as antiviral agents. Finally, the kinetics of processing in the nonstructural region was examined by pulse-chase analysis. NS3-containing precursors were absent, indicating that the 2/3 and 3/4A cleavages occur rapidly. In contrast, processing of the NS4A-5B region appeared to involve multiple pathways, and significant quantities of various polyprotein intermediates were observed. NS5B, the putative RNA polymerase, was found to be significantly less stable than the other mature cleavage products. This instability appeared to be an inherent property of NS5B and did not depend on expression of other viral polypeptides, including the HCV-encoded proteinases.     

Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene.

The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed.     

Assembly of turnip yellow mosaic virus replication complexes: interaction between the proteinase and polymerase domains of the replication proteins

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like supergroup, encodes two nonstructural replication proteins (140K and 66K), both of which are required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase activities, while the 66K protein encompasses the RNA-dependent RNA polymerase domain. Recruitment of the 66K protein to the sites of viral replication, located at the periphery of chloroplasts, is dependent upon the expression of the 140K protein. Using antibodies raised against the 140K and 66K proteins and confocal microscopy, we report the colocalization of the TYMV replication proteins at the periphery of chloroplasts in transfected or infected cells. The replication proteins cofractionated in functional replication complexes or with purified chloroplast envelope membranes prepared from infected plants. Using a two-hybrid system and coimmunoprecipitation experiments, we also provide evidence for a physical interaction of the TYMV replication proteins. In contrast to what has been found for other members of the alphavirus-like supergroup, the interaction domains were mapped to the proteinase domain of the 140K protein and to a large region encompassing the core polymerase domain within the 66K protein. Coexpression and colocalization experiments confirmed that the helicase domain of the 140K protein is unnecessary for the proper recruitment of the 66K protein to the chloroplast envelope, while the proteinase domain appears to be essential for that process. These results support a novel model for the interaction of TYMV replication proteins and suggest that viruses in the alphavirus-like supergroup may have selected different pathways to assemble their replication complexes.     

Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus.

The prophenoloxidase activating enzyme (ppA), a serine proteinase catalyzing the conversion of prophenoloxidase to an active phenoloxidase, has a molecular mass of about 36 kDa in its active form. This protein was cloned from a blood cell cDNA library and its corresponding cDNA of 1736 base pairs encodes a zymogenic protein (proppA) of 468 amino acids. An antibody raised against a synthetic peptide derived from a region of the cDNA sequence could efficiently inhibit the beta-1,3-glucan triggered activation of prophenoloxidase in vitro. The C-terminal half of the proppA is composed of a typical serine proteinase domain, with a sequence similar to other invertebrate and vertebrate serine proteinases. The N-terminal half contains a cationic glycine-rich domain, a cationic proline-rich domain and a clip-domain, in which the disulfide-bonding pattern is likely to be identical to those of the horseshoe crab big defensin and mammalian beta-defensins. Antibodies made against both the C- and the N-terminal halves recognize two proppAs under reducing conditions. However, under nonreducing conditions only the anti-C antibody recognized the two proppAs, which suggests that a conformational change takes place upon reduction that allows the anti-N to react with the N-terminal half of proppA. The recombinant clip-domain in crayfish proppA was overexpressed in Escherichia coli and the resulting peptide exhibited antibacterial activity against Gram-positive bacterial strains such as Micrococcus luteus Ml11 and Bacillus megaterium Bm11 with 50% growth inhibitory concentrations of 1.43 microM and 17.9 microM, respectively. These results suggest that the clip-domains in proppAs may function as antibacterial peptides.     

Functional analysis of AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis

The major peptidoglycan hydrolase of Enterococcus faecalis, AtlA, has been identified, but its enzyme activity remains unknown. We have used tandem mass spectrometry analysis of peptidoglycan hydrolysis products obtained using the purified protein to show that AtlA is an N-acetylglucosaminidase. To gain insight into the regulation of its enzyme activity, the three domains of AtlA were purified alone or in combination following expression of truncated forms of the atlA gene in Escherichia coli or partial digestion of AtlA by proteinase K. The central domain of AtlA was catalytically active, but its activity was more than two orders of magnitude lower than that of the complete protein. Partial proteolysis of AtlA was detected in vivo: zymograms of E. faecalis extracts revealed two catalytically active protein bands of 62 and 72 kDa that were both absent in extracts from an atlA null mutant. Limited digestion of AtlA by proteinase K in vitro suggested that the proteolytic cleavage of AtlA in E. faecalis extracts corresponds to the truncation of the N-terminal domain, which is rich in threonine and glutamic acid residues. We show that the truncation of the N-terminal domain from recombinant AtlA has no impact on enzyme activity. The C-terminal domain of the protein, which contains six LysM modules bound to highly purified peptidoglycan, was required for optimal enzyme activity. These data indicate that AtlA is not produced as a proenzyme and that control of the AtlA glucosaminidase activity is likely to occur at the level of LysM-mediated binding to peptidoglycan.     

Isolation, cloning and structural characterisation of boophilin, a multifunctional Kunitz-type proteinase inhibitor from the cattle tick

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine alpha-thrombin.boophilin complex, refined at 2.35 A resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S(1) pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9 degrees and is displaced by 6 A, while the C-terminal domain rotates almost 6 degrees accompanied by a 3 A displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1) residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.     

Crystal structure of the catalytic domain of DESC1, a new member of the type II transmembrane serine proteinase family

DESC1 was identified using gene-expression analysis between squamous cell carcinoma of the head and neck and normal tissue. It belongs to the type II transmembrane multidomain serine proteinases (TTSPs), an expanding family of serine proteinases, whose members are differentially expressed in several tissues. The biological role of these proteins is currently under investigation, although in some cases their participation in specific functions has been reported. This is the case for enteropeptidase, hepsin, matriptase and corin. Some members, including DESC1, are associated with cell differentiation and have been described as tumor markers. TTSPs belong to the type II transmembrane proteins that display, in addition to a C-terminal trypsin-like serine proteinase domain, a differing set of stem domains, a transmembrane segment and a short N-terminal cytoplasmic region. Based on sequence analysis, the TTSP family is subdivided into four subfamilies: hepsin/transmembrane proteinase, serine (TMPRSS); matriptase; corin; and the human airway trypsin (HAT)/HAT-like/DESC subfamily. Members of the hepsin and matriptase subfamilies are known structurally and here we present the crystal structure of DESC1 as a first member of the HAT/HAT-like/DESC subfamily in complex with benzamidine. The proteinase domain of DESC1 exhibits a trypsin-like serine proteinase fold with a thrombin-like S1 pocket, a urokinase-type plasminogen activator-type S2 pocket, to accept small residues, and an open hydrophobic S3/S4 cavity to accept large hydrophobic residues. The deduced substrate specificity for DESC1 differs markedly from that of other structurally known TTSPs. Based on surface analysis, we propose a rigid domain association for the N-terminal SEA domain with the back site of the proteinase domain.     

Stimulation of poly(A) polymerase through a direct interaction with the nuclear poly(A) binding protein allosterically regulated by RNA

During polyadenylation of mRNA precursors in metazoan cells, poly(A) polymerase is stimulated by the nuclear poly(A) binding protein PABPN1. We report that stimulation depends on binding of PABPN1 to the substrate RNA directly adjacent to poly(A) polymerase and results in an approximately 80-fold increase in the apparent affinity of poly(A) polymerase for RNA without significant effect on catalytic efficiency. PABPN1 associates directly with poly(A) polymerase either upon allosteric activation by oligo(A) or, in the absence of RNA, upon deletion of its N-terminal domain. The N-terminal domain of PABPN1 may function to inhibit undesirable interactions of the protein; the inhibition is relieved upon RNA binding. Tethering of poly(A) polymerase is mediated largely by the C-terminal domain of PABPN1 and is necessary but not sufficient for stimulation of the enzyme; an additional interaction dependent on a coiled-coil structure located within the N-terminal domain of PABPN1 is required for a productive interaction.     

Escherichia coli RNA polymerase subunit omega and its N-terminal domain bind full-length beta' to facilitate incorporation into the alpha2beta subassembly.

The omega subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (alpha2betabeta'). The presence of omega is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an omega-less strain of E. coli, GroEL appears to substitute for omega in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch on to beta' at its N-terminus and C-terminus. We show here that omega binds only the intact beta' subunit and not the beta' N-terminal domain or beta' C-terminal domain, implying that omega binding requires both these regions of beta'. We further show that omega can prevent the aggregation of beta' during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of omega is sufficient for binding and renaturation of beta'. CD and functional assays show that this N-terminal fragment retains the structure of native omega and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps alpha + alpha --> alpha2 + beta --> alpha2beta + beta' --> alpha2betabeta'. It is shown here that omega participates during the last stage of enzyme assembly when beta' associates with the alpha2beta subassembly.     

Amino acid residues critical for RNA-binding in the N-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells

The N-terminal domain of the coronavirus nucleocapsid (N) protein adopts a fold resembling a right hand with a flexible, positively charged β-hairpin and a hydrophobic palm. This domain was shown to interact with the genomic RNA for coronavirus infectious bronchitis virus (IBV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Based on its 3D structure, we used site-directed mutagenesis to identify residues essential for the RNA-binding activity of the IBV N protein and viral infectivity. Alanine substitution of either Arg-76 or Tyr-94 in the N-terminal domain of IBV N protein led to a significant decrease in its RNA-binding activity and a total loss of the infectivity of the viral RNA to Vero cells. In contrast, mutation of amino acid Gln-74 to an alanine, which does not affect the binding activity of the N-terminal domain, showed minimal, if any, detrimental effect on the infectivity of IBV. This study thus identifies residues critical for RNA binding on the nucleocapsid surface, and presents biochemical and genetic evidence that directly links the RNA binding capacity of the coronavirus N protein to the viral infectivity in cultured cells. This information would be useful in development of preventive and treatment approaches against coronavirus infection.     

Fate of membrane-bound reactants and products during the activation of human prothrombin by prothrombinase

Membrane binding by prothrombin, mediated by its N-terminal fragment 1 (F1) domain, plays an essential role in its proteolytic activation by prothrombinase. Thrombin is produced in two cleavage reactions. One at Arg(320) yields the proteinase meizothrombin that retains membrane binding properties. The second, at Arg(271), yields thrombin and severs covalent linkage with the N-terminal fragment 1.2 (F12) region. Covalent linkage with the membrane binding domain is also lost when prethrombin 2 (P2) and F12 are produced following initial cleavage at Arg(271). We show that at the physiological concentration of prothrombin, thrombin formation results in rapid release of the proteinase into solution. Product release from the surface can be explained by the weak interaction between the proteinase and F12 domains. In contrast, the zymogen intermediate P2, formed following cleavage at Arg(271), accumulates on the surface because of a approximately 20-fold higher affinity for F12. By kinetic studies, we show that this enhanced binding adequately explains the ability of unexpectedly low concentrations of F12 to greatly enhance the conversion of P2 to thrombin. Thus, the utilization of all three possible substrate species by prothrombinase is regulated by their ability to bind membranes regardless of whether covalent linkage to the F12 region is maintained. The product, thrombin, interacts with sufficiently poor affinity with F12 so that it is rapidly released from its site of production to participate in its numerous hemostatic functions.     

NMR structure of bacterial ribosomal protein l20: implications for ribosome assembly and translational control

L20 is a specific protein of the bacterial ribosome, which is involved in the early assembly steps of the 50S subunit and in the feedback control of the expression of its own gene. This dual function involves specific interactions with either the 23S rRNA or its messenger RNA. The solution structure of the free Aquifex aeolicus L20 has been solved. It is composed of an unstructured N-terminal domain comprising residues 1-58 and a C-terminal alpha-helical domain. This is in contrast with what is observed in the bacterial 50S subunit, where the N-terminal region folds as an elongated alpha-helical region. The solution structure of the C-terminal domain shows that several solvent-accessible, conserved residues are clustered on the surface of the molecule and are probably involved in RNA recognition. In vivo studies show that this domain is sufficient to repress the expression of the cistrons encoding L35 and L20 in the IF3 operon. The ability of L20 C-terminal domain to specifically recognise RNA suggests an assembly mechanism for L20 into the ribosome. The pre-folded C-terminal domain would make a primary interaction with a specific site on the 23S rRNA. The N-terminal domain would then fold within the ribosome, participating in its correct 3D assembly.