Sequence information within proteasomal prosequences mediates efficient integration of beta-subunits into the 20 S proteasome complex.

The maturation of proteases is governed by prosequences. During the biogenesis of the highly oligomeric eukaryotic 20 S proteasome five different prosequence-containing subunits have to be integrated and processed either by autocatalysis or by neighbouring subunits. To analyse the functional impact of proteasomal prosequences during complex formation, the propeptide of the facultative subunit beta1i/LMP2 was truncated to nine amino acid residues or completely deleted. Additionally, the charged residues within the truncated beta1i/LMP2 version were replaced by neutral residues. While deletion did not affect subunit incorporation, the presence of charged residues within the truncated version of the LMP2 propeptide diminished incorporation efficiency, an effect that was restored upon replacement of the charged amino acids against neutral components. During immunoproteasome formation, incorporation and processing of inducible proteasome beta-subunits are cooperative processes. We demonstrate a linear correlation of the levels of beta2i/MECL1 and beta1i/LMP2 within 20 S proteasomes, suggesting a physical interaction to be the molecular basis for the biased incorporation of both subunits. In the absence of beta5i/LMP7, precursor complexes containing unprocessed beta1i/LMP2 accumulated. The contribution of beta5i/LMP7 on the cooperative formation of a homogeneous population of immunoproteasome is therefore most likely based on an acceleration of the beta1i/LMP2 and potentially of beta2i/MECL1 processing kinetics.     

Mutations in the processing site of the precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit: effects on import,processing, assembly and stability

The small subunit (SSU) of Rubisco is synthesized in the cytosol in a precursor form. Upon import into the chloroplast, it is proteolytically processed at a Cys-Met bond to yield the mature form of the protein. To assess the importance of the Met residue for recognition and processing by the stromal peptidase, we substituted this residue with either Thr, Arg or Asp. The mutant precursor proteins were imported into isolated chloroplasts, and the products of the import reactions were analyzed. Mutants containing Thr or Arg residues at the putative processing site were processed to a single peptide, comigrating with the wild-type protein. N-terminal radio-sequencing revealed that these mutants were processed at the Cys-Thr and the Cys-Arg bond, respectively. After import of the Asp-containing mutant, four processed forms of the protein were observed. Analysis of the most abundant one, co-migrating with the wild-type protein, demonstrated that this species was also a product of correct processing, at the Cys-Asp bond. All the correctly processed peptides were found to be associated with the holoenzyme of Rubisco, and remained stable within the chloroplast, like the wild-type protein. The results of this study, together with previous ones, suggest that proper recognition and processing of the SSU precursor are more affected by residues N-terminal to the processing site than by the residue on the C-terminal side of this site.     

Novel propeptide function in 20 S proteasome assembly influences beta subunit composition

The assembly of eukaryotic 20 S proteasomes involves the formation of half-proteasomes where precursor beta-type subunits gather in position on an alpha-subunit ring, followed by the association of two half-proteasomes and beta-subunit processing. In vertebrates three additional beta-subunits (beta1i/LMP2, beta2i/MECL1, and beta5i/LMP7) can be synthesized and substituted for constitutive homologues (beta1/delta, beta2/Z, and beta5/X) to yield immunoproteasomes, which are important for generating certain antigenic peptides. We have shown previously that when all six beta-subunits are present, cooperative assembly mechanisms limit the diversity of proteasome populations. Specifically, LMP7 is incorporated preferentially over X into preproteasomes containing LMP2 and MECL1. We show here that the LMP7 propeptide is responsible for this preferential incorporation, and it also enables LMP7 to incorporate into proteasomes containing delta and Z. In contrast, the X propeptide restricts incorporation to proteasomes with delta and Z. Furthermore, we demonstrate that the LMP7 propeptide can function in trans when expressed on LMP2, and that its NH(2)-terminal and mid-regions are particularly critical for function. In addition to identifying a novel propeptide function, our results raise the possibility that one consequence of LMP7 incorporation into both immunoproteasomes and delta/Z proteasomes may be to increase the diversity of antigenic peptides that can be generated.     

Structural Insight into concerted inhibition of alpha 2 beta 2-type aspartate kinase from Corynebacterium glutamicum

Aspartate kinase (AK) catalyzes the first step of the biosynthesis of the aspartic acid family amino acids, and is regulated via feedback inhibition by end-products including Thr and Lys. To elucidate the mechanism of this inhibition, we determined the crystal structure of the regulatory subunit of AK from Corynebacterium glutamicum at 1.58 A resolution in the Thr-binding form, the first crystal structure of the regulatory subunit of alpha(2)beta(2)-type AK. The regulatory subunit contains two ACT domain motifs per monomer and is arranged as a dimer. Two non-equivalent ACT domains from different chains form an effector-binding unit that binds a single Thr molecule, and the resulting two effector-binding units of the dimer associate perpendicularly in a face-to-face manner. The regulatory subunit is a monomer in the absence of Thr but becomes a dimer by adding Thr. The dimerization is eliminated in mutant AKs with changes in the Thr-binding region, suggesting that the dimerization induced by Thr binding is a key step in the inhibitory mechanism of AK from C. glutamicum. A putative Lys-binding site and the inhibitory mechanism of CgAK are discussed.     

Identification of residues of the Saccharomyces cerevisiae G protein-coupled receptor contributing to alpha-factor pheromone binding

The Saccharomyces cerevisiae pheromone, alpha-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were studied as a model for peptide ligand-receptor interaction. The affinities and activities of various synthetic position-10 alpha-factor analogs with Ste2p expressing mutations at residues Ser47 and Thr48 were investigated. All mutant receptors were expressed at a similar level in the cytoplasmic membrane, and their efficacies of signal transduction were similar to that of the wild-type receptor. Mutant receptors differed in binding affinity (Kd) and potency (EC50) for gene induction by alpha-factor. One mutant receptor (S47K,T48K) had dramatically reduced affinity and activity for [Lys10]- and [Orn10]alpha-factor, whereas the affinity for Saccharomyces kluyveri alpha-factor (WHWLSFSKGEPMY) was increased over 20-fold compared with that of wild-type receptor. In contrast, the affinity of [Lys10]- and [Orn10]alpha-factor was increased greatly in a S47E,T48E mutant receptor, whereas the binding of the S. kluyveri alpha-factor was abolished. The affinity of [Lys10]- and [Orn10]alpha-factor for the S47E,T48E receptor dropped 4-6-fold in the presence of 1 m NaCl, whereas the affinity of alpha-factor was not affected by this treatment. These results demonstrate that when bound to its receptor the 10th residue (Gln) of the S. cerevisiae alpha-factor is adjacent to Ser47 and Thr48 residues in the receptor and that the 10th residue of alpha-factors from two Saccharomyces species is responsible for the ligand selectivity to their cognate receptors. Based on these data, we have developed a two-dimensional model of alpha-factor binding to its receptor.     

Characterisation of the newly identified human Ump1 homologue POMP and analysis of LMP7(beta 5i) incorporation into 20 S proteasomes

Biogenesis of mammalian 20 S proteasomes occurs via precursor complexes containing alpha and unprocessed beta subunits. A human homologue of the yeast proteasome maturation factor Ump1 was identified in 2D gels of 16 S precursor preparations and designated as POMP (proteasome maturation protein). We show that POMP is detected only in precursor fractions and not in fractions containing mature 20 S proteasome. Northern blot experiments revealed that expression of POMP is induced after treatment with interferon gamma. To analyse the role of the beta 5 propeptide for proper maturation and incorporation of the beta 5 subunit into the complex, human T2 cells, which highly express derivatives of the beta 5i subunit (LMP7), were studied. In contrast to yeast, the presence of the beta 5 propeptide is not essential for incorporation of LMP7 into the proteasome complex. Mutated LMP7 subunits either carrying the prosequence of beta 2i (LMP2) or containing a mutation in the active threonine site are incorporated like wild-type LMP7, while a LMP7 derivative lacking the prosequence completely is incorporated to a lesser extent. Although the absence of the prosequence does not affect incorporation of LMP7, its deletion leads to delayed proteasome maturation and thereby to an accumulation of precursor complexes. As a result of the precursor accumulation, an increased amount of the POMP protein can be detected in these cells.     

Substitution of ASP193 to ASN at the active site of ribulose-1,5-bisphosphate carboxylase results in conformational changes.

The crystal structure of a mutant of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillium rubrum, where Asp193, one of the ligands of the magnesium ion at the activator site, is replaced by Asn, has been determined to a nominal resolution of 0.26 nm. The mutation of Asp to Asn induces both local and global conformation changes as follows. The side chain of Asn193 moves away from the active site and interacts with main-chain oxygen of residue 165, located in the neighbouring strand beta 1 of the alpha/beta barrel. The side chain of Lys166, which forms a salt bridge with Asp193 in the wild-type enzyme, interacts with Asn54 from the second subunit and creates a new subunit-subunit interaction. Another new subunit-subunit interaction is formed, more than 1.2 nm away from the site of the mutation. In the mutant enzyme, the side chain of Asp263 interacts with the side chain of Thr106 from the second subunit. Asp193 is not part of a subunit-subunit interface area or an allosteric regulatory site. Nevertheless, replacement of this residue by Asn results, unexpectedly, in a difference in the packing of the two subunits, which can be described as a slight rotation of one of the subunits relative to the second. The observed structural changes at the active site of the enzyme provide a molecular explanation for the differing behaviour of the Asp193----Asn mutant with respect to activation.     

On the interaction of ribosomal protein L5 with 5S rRNA

Ribosomal protein L5, a 5S rRNA binding protein in the large subunit, is composed of a five-stranded antiparallel beta-sheet and four alpha-helices, and folds in a way that is topologically similar to the ribonucleprotein (RNP) domain [Nakashima et al., RNA 7, 692-701, 20011. The crystal structure of ribosomal protein L5 (BstL5) from Bacillus stearothermophilus suggests that a concave surface formed by an anti-parallel beta-sheet and long loop structures are strongly 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 occurred at beta-strands and loop structures in BstL5. The mutation of Lys33 at the beta 1-strand caused a significant reduction in 5S rRNA binding. In addition, the Arg92, Phe122, and Glu134 mutations on the beta2-strand, the alpha3-beta4 loop, and the beta4-beta5 loop, respectively, resulted in a moderate decrease in the 5S rRNA binding affinity. In contrast, mutation of the conserved residue Pro65 at the beta2-strand had little effect on the 5S rRNA binding activity. These results, taken together with previous results, identified Lys33, Asn37, Gln63, and Thr90 on the beta-sheet structure, and Phe77 at the beta2-beta3 loop as critical residues for the 5S rRNA binding. The contribution of these amino acids to 5S rRNA binding was further quantitatively evaluated by surface plasmon resonance (SPR) analysis by the use of BIAcore. The results showed that the amino acids on the beta-sheet structure are required to decrease the dissociation rate constant for the BstL5-5S rRNA complex, while those on the loops are to increase the association rate constant for the BstL5-5S rRNA interaction.     

Charged residues in the beta2 subunit involved in GABAA receptor activation

Fast synaptic inhibition in the mammalian central nervous system is mediated primarily via activation of the gamma-aminobutyric acid type A receptor (GABAA-R). Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state. This transition, known as gating, is thought to be associated with a sequence of conformational changes originating at the agonist-binding site, ultimately resulting in opening of the channel. Using site-directed mutagenesis and several different GABAA-R agonists, we identified a number of highly conserved charged residues in the GABAA-R beta2 subunit that appear to be involved in receptor activation. We then used charge reversal double mutants and disulfide trapping to investigate the interactions between these flexible loops within the beta2 subunit. The results suggest that interactions between an acidic residue in loop 7 (Asp146) and a basic residue in pre-transmembrane domain-1 (Lys215) are involved in coupling agonist binding to channel gating.     

Direct identification of lysine-33 as the principal cationic center of the omega-amino acid binding site of the recombinant kringle 2 domain of tissue-type plasminogen activator.

We have generated site-specific mutants of the kringle 2 domain of tissue-type plasminogen activator [( K2tPA]) in order to identify directly the cationic center of the protein that is responsible for its interaction with the carboxyl group of important omega-amino acid effector molecules, such as epsilon-amino caproic acid (EACA). Molecular modeling of [K2tPA], docked with EACA, based on crystal structures of the kringle 2 region of prothrombin and the kringle 4 domain of human plasminogen, clearly shows that Lys33 is the only positively charged amino acid in [K2tPA] that is sufficiently proximal to the carboxyl group of the ligand to stabilize this interaction. In order to examine directly the importance of this particular amino acid residue in this interaction, we have constructed, expressed, and purified three recombinant (r) mutants of [K2tPA], viz., Lys33Thr, Lys33Leu, and Lys33Arg, and found that only the last variant retained significant ability to interact with EACA and several of its structural analogues at neutral pH. In addition, another mutated r-[K2tPA], i.e., Lys33His, interacts very weakly with omega-amino acids at neutral pH and much more strongly at lower pH values where His33 would be expected to undergo protonation. This demonstrates that any positively charged amino acid at position 33 satisfies the requirement for mediation of significant bindings to this class of molecules. Since, in other kringles, positively charged residues at amino acid sequence positions homologous to Lys68, Arg70, and Arg71 of [K2tPA] have been found to participate in kringle interactions with EACA-like compounds, we have also examined the binding of EACA, and some of its analogues, to three additional r-[K2tPA] variants, i.e., Lys68Ala, Arg70Ala, and Arg71Ala. In each case, binding of these omega-amino acids to the variant kringles was observed, with only the Lys68Ala variant showing a slightly diminished capacity for this interaction. These investigations provide clear and direct evidence that Lys33 is the principal cationic site in wild-type r-[K2tPA] that directly interacts with the carboxyl group of omega-amino acid effector molecules.     

Sequence requirements for precursor cleavage within the constitutive secretory pathway.

We have recently demonstrated that the Arg-X-Lys/Arg-Arg sequence is a signal for precursor cleavage catalyzed by furin, a mammalian homologue of the yeast precursor-processing endoprotease Kex2, within the constitutive secretory pathway. In this study, we further examined sequence requirements for the constitutive precursor cleavage by expression of various prorenin mutants with amino acid substitutions around the native Lys-Arg cleavage site in Chinese hamster ovary cells. The results delineate the following sequence rules that govern the constitutive precursor cleavage. (a) A basic residue (Lys or Arg) at the 4th (position -4) or 6th (position -6) residue upstream of the cleavage site besides basic residues at positions -1 and -2 is necessary. (b) At position -2, a Lys residue is more preferable than Arg. (c) At position -4, an Arg residue is more preferable than Lys. (d) At position 1, a hydrophobic aliphatic amino acid is not suitable.     

Polypeptide substrate specificity of PsLSMT. A set domain protein methyltransferase

Rubisco large subunit methyltransferase (PsLSMT) is a SET domain protein responsible for the trimethylation of Lys-14 in the large subunit of Rubisco. The polypeptide substrate specificity determinants for pea Rubisco large subunit methyltransferase were investigated using a fusion protein construct between the first 23 amino acids from the large subunit of Rubisco and human carbonic anhydrase II. A total of 40 conservative and non-conservative amino acid substitutions flanking the target Lys-14 methylation site (positions P(-3) to P(+3)) were engineered in the fusion protein. The catalytic efficiency (k(cat)/K(m)) of PsLSMT was determined using each of the substitutions and a polypeptide consensus recognition sequence deduced from the results. The consensus sequence, represented by X-(Gly/Ser)-(Phe/Tyr)-Lys-(Ala/Lys/Arg)-(Gly/Ser)-pi, where X is any residue, Lys is the methylation site, and pi is any aromatic or hydrophobic residue, was used to predict potential alternative substrates for PsLSMT. Four chloroplast-localized proteins were identified including gamma-tocopherol methyltransferase (gamma-TMT). In vitro methylation assays using PsLSMT and a bacterially expressed form of gamma-TMT from Perilla frutescens confirmed recognition and methylation of gamma-TMT by PsLSMT in vitro. RNA interference-mediated knockdown of the PsLSMT homologue (NtLSMT) in transgenic tobacco plants resulted in a 2-fold decrease of alpha-tocopherol, the product of gamma-TMT. The results demonstrate the efficacy of consensus sequence-driven identification of alternative substrates for PsLSMT as well as identification of functional attributes of protein methylation catalyzed by LSMT.     

Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha2beta2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens. We converted 16 residues in the human alpha subunit individually to other amino acids, and expressed the mutant polypeptides together with the wild-type beta subunit in insect cells. Asp414Ala and Asp414Asn inactivated the enzyme completely, whereas Asp414Glu increased the K(m) for Fe2+ 15-fold and that for 2-oxoglutarate 5-fold. His412Glu, His483Glu and His483Arg inactivated the tetramer completely, as did Lys493Ala and Lys493His, whereas Lys493Arg increased the K(m) for 2-oxoglutarate 15-fold. His501Arg, His501Lys, His501Asn and His501Gln reduced the enzyme activity by 85-95%; all these mutations increased the K(m) for 2-oxoglutarate 2- to 3-fold and enhanced the rate of uncoupled decarboxylation of 2-oxoglutarate as a percentage of the rate of the complete reaction up to 12-fold. These and other data indicate that His412, Asp414 and His483 provide the three ligands required for the binding of Fe2+ to a catalytic site, while Lys493 provides the residue required for binding of the C-5 carboxyl group of 2-oxoglutarate. His501 is an additional critical residue at the catalytic site, probably being involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate and the decarboxylation of this cosubstrate.     

Familial Alzheimer disease presenilin-1 mutations alter the active site conformation of γ-secretase

Presenilin-1 (PS1) is the catalytic subunit of γ-secretase, and mutations in this protein cause familial Alzheimer Disease (FAD). However, little is known about how these mutations affect the active site of γ-secretase. Here, we show that PS1 mutations alter the S2 subsite within the active site of γ-secretase using a multiple photoaffinity probe approach called "photophore walking." Moreover, we developed a unique in vitro assay with a biotinylated recombinant Notch1 substrate and demonstrated that PS1 FAD mutations directly and significantly reduced γ-secretase activity for Notch1 cleavage. Substitution of the Notch Cys-1752 residue, which interacts with the S2 subsite, with Val, Met, or Ile has little effect on wild-type PS1 but leads to more efficient substrates for mutant PS1s. This study indicates that alteration of this S2 subsite plays an important role in determining the activity and specificity of γ-secretase for APP and Notch1 processing, which provides structural basis and insights on how certain PS1 FAD mutations lead to AD pathogenesis.     

Human p53 inhibits growth in Schizosaccharomyces pombe.

Overexpression of wild-type p53 in mammalian cells blocks growth. We show here that the overexpression of wild-type human p53 in the fission yeast Schizosaccharomyces pombe also blocks growth, whereas the overexpression of mutant forms of p53 does not. The p53 polypeptide is located in the nucleus and is phosphorylated at both the cdc2 site and the casein kinase II site in S. pombe. A new dominant mutation of p53, resulting in the change of a cysteine to an arginine at amino acid residue 141, was identified. The results presented here demonstrate that S. pombe could provide a simple system for studying the mechanism of action of human p53.     

Structural basis for the inactivation of AdoMetDC K12R mutant

S-adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine. Polyamines are ubiquitous organic cations that are absolutely required for normal cell proliferation and differentiation. AdoMetDC catalyzes decarboxylation of S-adenosylmethionine (AdoMet) which provides aminopropyl groups for spermidine and spermine synthesis. Mammalian AdoMetDC is produced as a proenzyme (38 kDa) which is cleaved to form the alpha (30.7 kDa) and beta (7.7 kDa) subunits of the mature enzyme. It is here shown that the catalytic activity of the enzyme was completely eliminated when lysine 12 was mutated to an arginine residue in the small subunit; however, the proenzyme processing was not affected. On the other hand, mutations of other lysine residues (Lys45-->Arg and Lys56-->Arg) did not affect either the enzyme activity or the proenzyme processing. Structure analysis using Swiss Deep Viewer v3.7 has indicated that Arg in place of Lys12 may eliminate AdoMetDC activity by restricting the mobility of Thr85 through hydrogen bonding. Sequence alignment of various AdoMetDC sequences indicated that Thr85 is in a highly conserved region, suggesting that Thr85 is critical for the decarboxylation reaction.     

An attenuating mutation in nsP1 of the Sindbis-group virus S.A.AR86 accelerates nonstructural protein processing and up-regulates viral 26S RNA synthesis

The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.     

Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: use of Mn2+ to show interaction of serine 16 with Mg2+.

MgATP-binding and hydrolysis are an integral part of the nitrogenase catalytic mechanism. We are exploring the function of MgATP hydrolysis in this reaction by analyzing the properties of the Fe protein (FeP) component of Azotobacter vinelandii nitrogenase altered by site-directed mutagenesis. We have previously (Seefeldt, L.C., Morgan, T.V., Dean, D.R., & Mortenson, L.E., 1992, J. Biol. Chem. 267, 6680-6688) identified a region near the N-terminus of FeP that is involved in interaction with MgATP. This region of FeP is homologous to the well-known nucleotide-binding motif GXXXXGKS/T. In the present work, we examined the function of the four hydroxyl-containing amino acids immediately C-terminal to the conserved lysine 15 that is involved in interaction with the gamma-phosphate of MgATP. We have established, by altering independently Thr 17, Thr 18, and Thr 19 to alanine, that a hydroxyl-containing residue is not needed at these positions for FeP to function. In contrast, an hydroxyl-containing amino acid at position 16 was found to be critical for FeP function. When the strictly conserved Ser 16 was altered to Ala, Cys, Asp, or Gly, the FeP did not support N2 fixation when expressed in place of the wild-type FeP in A. vinelandii. Altering Ser 16 to Thr (S16T), however, resulted in the expression of an FeP that was partially active. This S16T FeP was purified to homogeneity, and its biochemical examination allowed us to assign a catalytic function to this hydroxyl group in the nitrogenase mechanism. Of particular importance was the finding that the S16T FeP had a significantly higher affinity for MgATP than the wild-type FeP, with a measured Km of 20 microM compared to the wild-type FeP Km of 220 microM. This increased kinetic affinity for MgATP was reflected in a significantly stronger binding of the S16T FeP for MgATP. In contrast, the affinity for MgADP, which binds at the same site as MgATP, was unchanged. The catalytic efficiency (kcat/Km) of S16T FeP was found to be 5.3-fold higher than for the wild-type FeP, with the S16T FeP supporting up to 10 times greater nitrogenase activity at low MgATP concentrations. This indicates a role for the hydroxyl group at position 16 in interaction with MgATP but not MgADP. The site of interaction of this residue was further defined by examining the properties of wild-type and S16T FePs in utilizing MnATP compared with MgATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multiple pocket recognition of SNAP25 by botulinum neurotoxin serotype E

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