NADP-Dependent enzymes. I: Conserved stereochemistry of cofactor binding

The ubiquitous redox cofactors nicotinamide adenine dinucleotides [NAD and NADP] are very similar molecules, despite their participation in substantially different biochemical processes. NADP differs from NAD in only the presence of an additional phosphate group esterified to the 2′-hydroxyl group of the ribose at the adenine end and yet NADP is confined with few exceptions to the reactions of reductive biosynthesis, whereas NAD is used almost exclusively in oxidative degradations. The discrimination between NAD and NADP is therefore an impressive example of the power of molecular recognition by proteins. The many known tertiary structures of NADP complexes affords the possibility for an analysis of their discrimination. A systematic analysis of several crystal structures of NAD(P)-protein complexes show that: 1) the NADP coenzymes are more flexible in conformation than those of NAD; 2) although the protein-cofactor interactions are largely conserved in the NAD complexes, they are quite variable in those of NADP; and 3) in both cases the pocket around the nicotinamide moiety is substrate dependent. The conserved and variable interactions between protein and cofactors in the respective binding pockets are reported in detail. Discrimination between NAD and NADP is essentially a consequence of the overall pocket and not of a few residues. A clear fingerprint in NAD complexes is a carboxylate side chain that chelates the diol group at the ribose near the adenine, whereas in NADP complexes an arginine side chain faces the adenine plane and interacts with the phosphomonoester. The latter type of interaction might be a general feature of recognition of nucleotides by proteins. Other features such as strand-like hydrogen bonding between the NADP diphosphate moeties and the protein are also significant. The NADP binding pocket properties should prove useful in protein engineering and design. © 1997 Wiley-Liss Inc.     

Three-dimensional structure of horse liver alcohol dehydrogenase at 2-4 A resolution.

The crystal structure analysis of horse liver alcohol dehydrogenase has been extended to 2.4 Å resolution. From the corresponding electron density map of the apoenzyme we have determined the positions of the 374 amino acids in the polypeptide chain of each subunit.The coenzyme binding domain of the subunit comprises residues 176 to 318. 45% of these residues are helical and 32% are in the central six-stranded pleated sheet structure. The positions and orientations of the helices with respect to the pleated sheet indicate a possible folding mechanism for this part of the subunit structure. The coenzyme analogue ADP-ribose binds to this domain in a position and orientation very similar to coenzyme binding to lactate dehydrogenase. The adenine part binds in a hydrophobic pocket, the adenosine ribose is hydrogen-bonded to the side chain of Asp223, the pyrophosphate is positioned by interaction with Arg47 and the nicotinamide ribose is 6Å away from the catalytic zinc atom.The catalytic domain is mainly built up from three distinct antiparallel pleated-sheet regions. Residues within this domain provide ligands to the catalytic zinc atom; Cys46, His67 and Cys174. An approximate tetrahedral coordination of this zinc is completed by a water molecule or hydroxyl ion depending on the pH. Residues 95 to 113 form a lobe that binds the second zinc atom of the subunit. This zinc is liganded in a distorted tetrahedral arrangement by four sulphur atoms from the cysteine residues 97, 100, 103 and 111. The lobe forms one side of a significant cleft in the enzyme surface suggesting that this region might constitute a second catalytic centre of unknown function.The two domains of the subunit are separated by a crevice that contains a wide and deep hydrophobic pocket. The catalytic zinc atom is at the bottom of this pocket, with the zinc-bound water molecule projecting out into the pocket. This water molecule is hydrogen-bonded to the side chain of Ser48 which in turn is hydrogen-bonded to His51. The pocket which in all probability is the binding site for the substrate and the nicotinamide moiety of the coenzyme, is lined almost exclusively with hydrophobic side chains. Both subunits contribute residues to each of the two substrate binding pockets of the molecule. The only accessible polar groups in the vicinity of the catalytic centre are Ser48 and Thr178 apart from zinc and the zinc-bound water molecule.     

Mutational replacements in subtilisin 309. Val104 has a modulating effect on the P4 substrate preference.

The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has been investigated. The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phe, Ser, Trp and Tyr by site-directed mutagenesis. It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position-104 residue. With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates. The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate-induced conformational change. However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket. The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4.     

The specificity of interaction between S-adenosyl-L-methionine and a nucleolar 2'-O-methyltransferase

The structural features of S-adenosyl-L-methionine (SAM)3 required for optimal binding to a nucleolar 2'-O-methyltransferase were elucidated using various analogs of SAM with modifications of the amino acid, sugar, sulfonium center, and base portions of the molecule. Equilibrium binding constants for SAM and each analog were determined by a nitrocellulose filter binding assay. To ensure the chiral and chemical purity of the 3H-labeled SAM used in the binding experiments, a cation-exchange HPLC procedure was developed to separate degradation products of SAM such as adenine and 5'-deoxy-5'-methylthioadenosine, as well as to separate the (S,S)-SAM from the biologically inactive (R,S)-SAM stereoisomer. Results from these studies demonstrated that S-adenosyl-L-homocysteine, a product of the methyltransferase reaction, bound equally as well as (S,S)-SAM, indicating that neither the charge nor the methyl group at the sulfonium center of (S,S)-SAM is essential for maximal binding. Other modifications of the sulfonium center demonstrated that a sulfur to carbon atom replacement had little effect on binding affinity, whereas substituting an ethyl group for the methyl group greatly reduced the binding affinity. In addition, the chirality at the sulfonium center was important. The naturally occurring S-chiral form had a 10-fold higher binding affinity than the R-chiral form. No significant stereospecificity was observed relative to the chiral alpha-carbon of the methionine moiety in SAM. The alpha-amino group of methionine and the 6-amino group of adenine were both required for maximal binding, while the loss of the 2'-hydroxyl group on the ribose moiety was not. Taken together, these results defined some of the specific geometric and functional group requirements which affect the specificity of interaction between S-adenosyl-L-methionine and the nucleolar 2'-O-methyltransferase.     

RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids

The RNA-binding/dimerization domain of the NS1 protein of influenza A virus (73 amino acids in length) exhibits a novel dimeric six-helical fold. It is not known how this domain binds to its specific RNA targets, one of which is double-stranded RNA. To elucidate the mode of RNA binding, we introduced single alanine replacements into the NS1 RNA-binding domain at specific positions in the three-dimensional structure. Our results indicate that the dimer structure is essential for RNA binding, because any alanine replacement that causes disruption of the dimer also leads to the loss of RNA-binding activity. Surprisingly, the arginine side chain at position 38, which is in the second helix of each monomer, is the only amino-acid side chain that is absolutely required only for RNA binding and not for dimerization, indicating that this side chain probably interacts directly with the RNA target. This interaction is primarily electrostatic, because replacement of this arginine with lysine had no effect on RNA binding. A second basic amino acid, the lysine at position 41, which is also in helix 2, makes a strong contribution to the affinity of binding. We conclude that helix 2 and helix 2', which are antiparallel and next to each other in the dimer conformation, constitute the interaction face between the NS1 RNA-binding domain and its RNA targets, and that the arginine side chain at position 38 and possibly the lysine side chain at position 41 in each of these antiparallel helices contact the phosphate backbone of the RNA target.     

Chromogenic substrates of bovine beta-trypsin: the influence of an amino acid residue in P1 position on their interaction with the enzyme

The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design and synthesize a series of trypsin chromogenic substrates modified in position P1: Ac-Ala-Val-Abu-Pro-X-pNA, where X = Orn, Lys, Arg, Har, Arg(NO(2)), Cit, Hci, Phe(p-CN), Phe(p-NH(2)); pNA = p-nitroanilide. The most active compounds (as determined by specificity constant k(cat)/K(m)) were peptides with the Arg and Lys residues in the position discussed. Changes in the length and the decrease of the positive charge of the amino acid residue side chain in position P(1) resulted in the decrease or loss of the affinity towards bovine beta-trypsin. Among peptides containing amino acid residues with uncharged side chains in position P1, only one with p-cyano-l-Phe revealed activity. These results correspond well with trypsin inhibitory activity of CMTI-III analogues modified in the equivalent position, indicating the same type of interaction between position P1 of the substrate or inhibitor and S1 site specificity of trypsin.     

Crosslinking of tRNAs by the carcinostatic agent dirhodium tetraacetate

A crystalline complex of yeast tRNA(phe) and dirhodium tetraacetate (DRTA) was prepared and its X-ray structure determined. The bifunctional DRTA forms an intermolecular cross-link between the N(1) position of adenine A36 in the anticodon triplet and possibly a ribose hydroxyl group of residue A76 at the 3' terminus of a symmetry related tRNA molecule. The rhodium complex apparently shows a preference for binding to the N(1) position of adenine in a single strand region of the tRNA molecule.     

Site-directed analysis of the functional domains in the factor Xa inhibitor tick anticoagulant peptide: identification of two distinct regions that constitute the enzyme recognition sites.

Recombinant tick anticoagulant peptide (rTAP) is a highly selective inhibitor of blood coagulation factor Xa. rTAP has been characterized kinetically as a slow, tight-binding, competitive inhibitor of the enzyme. We used an approach consisting of both recombinant, site-directed mutagenesis and solid-phase chemical synthesis to generate 31 independent mutations in rTAP to identify those regions of the molecule which contribute to the specific, high-affinity binding interaction with factor Xa. Our results demonstrate that the four amino-terminal residues of rTAP constitute the primary recognition determinant necessary for the formation of the high-affinity enzyme-inhibitor complex. The Arg residue in position three is probably not interacting with the S1-specificity pocket of factor Xa in a substrate-like manner since substitution at this position with a D-Arg amino acid produced only a modest decrease in affinity (5-fold). An additional domain in the rTAP molecule located between residues 40 and 54 was identified as a probable secondary binding determinant. Interestingly, this region in rTAP shares significant amino acid sequence homology with a sequence in prothrombin immediately amino-terminal to the factor Xa cleavage site that generates meizothrombin. These observations indicate that specific segments within two different regions of the rTAP molecule contribute to the potent binding interaction between rTAP and factor Xa.     

Extended repertoire of permissible peptide ligands for HLA-B*2702.

Recognition of self peptides bound to the class I major histocompatibility complex molecule HLA-B27 is thought to trigger proliferation of autoreactive T cells and result in autoimmune arthritic diseases. Previous work from other laboratories established that a predominant feature of endogenous peptides eluted from purified B27 is an arginine at position 2. We studied the binding of peptides containing both natural and unnatural amino acids by the subtype HLA-B*2702, with the goal of gaining insight into peptide binding by this B27 subtype that is associated with susceptibility to arthritic disease. A soluble from of B*2702 was depleted of endogenous peptides. We tested the binding of peptides substituted with cysteine, homocysteine, or an alpha-amino-epsilon-mercapto hexanoic acid side chain (Amh) instead of the naturally occurring arginine at position 2, to determine whether the peptide sulfhydryl residue could be covalently linked to cysteine 67 in the B*2702 binding cleft. Although none of the altered peptide sequences bound covalently to B*2702, the affinities of the homocysteine- and Amh-substituted peptides were close to that of the native peptide sequence. Substitutions at position 2 with other side chains, such as glutamine and methionine, also resulted in peptides that bound with only slightly reduced affinity. These results demonstrate that peptide side chains other than arginine at position 2 can be accomodated within the B*2702 peptide binding site with only minor reductions in affinity. This extended repertoire of permissible B27-binding peptides should be taken into account for a consideration of disease-associated peptide sequences.     

Crystal structure of the anticarcinogenic Bowman-Birk inhibitor from snail medic (Medicago scutellata) seeds complexed with bovine trypsin

The structure of the ternary complex of the anticarcinogenic Bowman-Birk protease inhibitor purified from snail medic (Medicago scutellata) seeds (MSTI) and two molecules of bovine trypsin has been solved by X-ray diffraction analysis of single crystals to a resolution of 2.0 A. This is the highest resolution model of a ternary complex of this type currently available. The two binding loops of the MSTI differ in only one amino acid and have in both cases an arginine in position P1. The distances between the residues of the inhibitor at the binding interface and the trypsin side chains that recognize them are almost identical in the two sites. When compared to the NMR model of the uncomplexed MSTI, the inhibitor in the functional assembly with trypsin shows the largest differences in the two P2' residues. Compared with the similar ternary complex of the soybean trypsin inhibitor, this model shows very small differences in the polypeptide chain of the trypsin binding sites and its largest difference in the area between Asp 26 and His 32 of the MSTI which in the soybean inhibitor has an extra Leu inserted in position 29.     

5'-bromoacetamido-5'-deoxyadenosine. A novel reagent for labeling adenine nucleotide sites in proteins

We have synthesized and characterized 5'-bromoacetamido-5'-deoxyadenosine (5'-BADA), a new reagent for labeling adenine nucleotide binding sites in enzymatic and regulatory proteins. 5'-BADA possessed exceptionally high solubility and stability in aqueous buffers between pH 5.0 and 8.6 at 25 degrees C. A Dixon plot of data from enzyme kinetic measurements showed that 5'-BADA is a competitive inhibitor of NADH oxidation by 3 alpha,20 beta-hydroxysteroid dehydrogenase with a Ki value of 11.8 mM. This compares with a Ki value of 10 mM for adenosine under similar experimental conditions. Incubating 5'-BADA with a 3 alpha,20 beta-hydroxysteroid dehydrogenase at pH 7.0 and 25 degrees C caused simultaneous loss of both 3 alpha and 20 beta activity. The enzyme inactivation reaction proceeded by a first order kinetic process. The rates of enzyme inactivation as a function of 5'-BADA concentration obeyed saturation kinetics. 2-Bromoacetamide, at ten times the maximum concentration of 5'-BADA, had no measurable effect on enzyme activity during 25 h of incubation. NADH and AMP protected 3 alpha,20 beta-hydroxysteroid dehydrogenase against inactivation by 5'-BADA. The results suggest that 5'-BADA inactivates the enzyme by irreversibly binding to the adenine domain of the NADH cofactor binding region at the catalytic site of 3 alpha,20 beta-hydroxysteroid dehydrogenase. Irreversible binding follows from an alkylation reaction between the bromoacetamido side chain of 5'-BADA and an amino acid at or near the enzyme catalytic site. 5'-BADA is presented as a new reagent for selectively labeling amino acid residues at the adenine nucleotide binding sites of enzymatic and regulatory proteins.     

On the mechanism of cholesterol interaction with apolipoproteins A-I and E.

It is shown that cholesterol may interact with some substances containing the guanidine group (guanidine itself, arginine, metformin and dodecylguanidine bromide) and with arginine-rich proteins--apoproteins A-I and E. In the latter case the interaction produces the formation of cholesterol-apoprotein complexes. Analysis of such complexes has shown that one apo A-I molecule binds 17-22 and one apo E molecule binds 30-35 sterol molecules, which approximately corresponds to the amount of arginine residues in these proteins. Formation of cholesterol-apoprotein complexes has been suggested to occur due to: (1) formation of hydrogen bond and/or ion-dipole interaction between cholesterol hydroxyl and guanidine groups of the apoprotein arginine residues and (2) hydrophobic interaction of the cholesterol aliphatic chain with nonpolar side chains of the amino acids occupying the third position from arginine in the protein molecule.     

Crosslinking of tRNAs by the Carcinostatic Agent Dirhodium Tetraacetate

Abstract

A crystalline complex of yeast tRNA^phe and dirhodium tetraacetate (DRTA) was prepared and its X-ray structure determined. The bifunctional DRTA forms an intermolecular crosslink between the N(1) position of adenine A36 in the anticodon triplet and possibly a ribose hydroxyl group of residue A76 at the 3′ terminus of a symmetry related tRNA molecule. The rhodium complex apparently shows a preference for binding to the N(l) position of adenine in a single strand region of the tRNA molecule.     

Glycine oxidase from Bacillus subtilis. Characterization of a new flavoprotein.

Glycine oxidase (GO) is a homotetrameric flavoenzyme that contains one molecule of non-covalently bound flavin adenine dinucleotide per 47 kDa protein monomer. GO is active on various amines (sarcosine, N-ethylglycine, glycine) and d-amino acids (d-alanine, d-proline). The products of GO reaction with various substrates have been determined, and it has been clearly shown that GO catalyzes the oxidative deamination of primary and secondary amines, a reaction similar to that of d-amino acid oxidase, although its sequence homology is higher with enzymes such as sarcosine oxidase and N-methyltryptophane oxidase. GO shows properties that are characteristic of the oxidase class of flavoproteins: it stabilizes the anionic flavin semiquinone and forms a reversible covalent flavin-sulfite complex. The approximately 300 mV separation between the two FAD redox potentials is in accordance with the high amount of the anionic semiquinone formed on photoreduction. GO can be distinguished from d-amino acid oxidase by its low catalytic efficiency and high apparent K(m) value for d-alanine. A number of active site ligands have been identified; the tightest binding is observed with glycolate, which acts as a competitive inhibitor with respect to sarcosine. The presence of a carboxylic group and an amino group on the substrate molecule is not mandatory for binding and catalysis.     

Uridine(5')diphospho(1)-alpha-D-glucose. A binding study to glycogen phosphorylase b in the crystal

UDP-glucose is an R-state inhibitor of glycogen phosphorylase b, competitive with the substrate, glucose 1-phosphate and noncompetitive with the allosteric activator, AMP. Diffusion of 100 mM UDP-glucose into crystals of phosphorylase b resulted in a difference Fourier synthesis at 0.3-nm resolution that showed two peaks: (a) binding at the allosteric site and (b) binding at the catalytic site. At the allosteric site the whole of the UDP-glucose molecule can be located. It is in a well defined folded conformation with its uracil portion in a similar position to that observed for the adenine of AMP. The uracil and the glucose moieties stack against the aromatic side chains of Tyr-75 and Phe-196, respectively. The phosphates of the pyrophosphate component interact with Arg-242, Arg-309 and Arg-310. At the catalytic site, the glucose-1-P component of UDP-glucose is firmly bound in a position similar to that observed for glucose 1-phosphate. The pyrophosphate is also well located with the glucose phosphate interacting with the main-chain NH groups at the start of the glycine-loop alpha helix and the uridine phosphate interacting through a water molecule with the 5'-phosphate of the cofactor pyridoxal phosphate and with the side chains of residues Tyr-573, Lys-574 and probably Arg-569. However the position of the uridine cannot be located although analysis by thin-layer chromatography showed that no degradation had taken place. Binding of UDP-glucose to the catalytic site promotes extensive conformational changes. The loop 279-288 which links the catalytic site to the nucleoside inhibitor site is displaced and becomes mobile. Concomitant movements of residues His-571, Arg-569, and the loop 378-383, together with the major loop displacement, result in an open channel to the catalytic site. Comparison with other structural results shows that these changes form an essential feature of the T to R transition. They allow formation of the phosphate recognition site at the catalytic site and destroy the nucleoside inhibitor site. Kinetic experiments demonstrate that UDP-glucose activates the enzyme in the presence of high concentrations of the weak activator IMP, because of its ability to decrease the affinity of IMP for the inhibitor site.     

Major histocompatibility complex class II binding characteristics of peptoid-peptide hybrids

The major histocompatibility complex (MHC) class II binding requirements for solvent-exposed peptide residues were systematically studied using amino acid and peptoid substitutions. In a peptoid residue, the side chain is present on the backbone nitrogen atom as opposed to the alpha-carbon atom in an amino acid residue. To investigate the effect of this side chain shifting on MHC binding, three amino acids in the central part of the peptide sticking out of the binding groove were replaced by corresponding peptoid residues. Two peptoid-peptide hybrids showed large affinity decreases in the MHC-peptide binding assay. To investigate this affinity loss, the individual contributions to MHC binding affinity of the side chain (position), the putative hydrogen bond, and the flexibility were dissected. We conclude that the side chain position as well as the backbone nitrogen atom hydrogen bonding features of solvent-exposed residues in the peptide can be important for MHC binding affinity.     

Comparative molecular model building of two serine proteinases from cytotoxic T lymphocytes

Two genes that are expressed when precursor cytotoxic T lymphocytes are transformed to T killer cells have been cloned and sequenced. The derived amino acid sequences, coding for cytotoxic cell protease 1 (CCP1) and Hannuka factor (HF) are highly homologous to members of the serine proteinase family. Comparative molecular model building using the known three-dimensional structures and the derived amino acid sequences of the lymphocyte enzymes has provided useful structural information, especially in predicting the conformations of the substrate binding sites. In applying this modelling procedure, we used the X-ray structures of four serine proteinases to provide a structurally based sequence alignment: alpha-chymotrypsin (CHT), bovine trypsin (BT), Streptomyces griseus trypsin (SGT), and rat mast cell protease 2 (RMCP2). The root mean square differences in alpha-carbon atom positions among these four structures when compared in a pairwise fashion range from 0.79 to 0.97 A for structurally equivalent residues. The sequences of the two lymphocyte enzymes were then aligned to these proteinases using chemical criteria and the superimposed X-ray structures as guides. The alignment showed that the sequence of CCP1 was most similar to RMCP2, whereas HF has regions of homology with both RMCP2 and BT. With RMCP2 as a template for CCP1 and the two enzymes RMCP2 and BT as templates for HF, the molecular models were constructed. Intramolecular steric clashes that resulted from the replacement of amino acid side chains of the templates by the aligned residues of CCP1 and HF were relieved by adjustment of the side chain conformational angles in an interactive computer graphics device. This process was followed by energy minimization of the enzyme model to optimize the stereochemical geometry and to relieve any remaining unacceptably close nonbonded contacts. The resulting model of CCP1 has an arginine residue at position 226 in the specificity pocket, thereby predicting a substrate preference for P1 aspartate or glutamate residues. The model also predicts favorable binding for a small hydrophobic residue at the P2 position of the substrate. The primary specificity pocket of HF resembles that of BT and therefore predicts a lysine or arginine preference for the P1 residue. The arginine at position 99 in the model of HF suggests a preference for aspartate or glutamate side chains in the P2 position of the substrate. Both CCP1 and HF have a free cysteine in the segment of polypeptide 88 to 93.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specificity of the basic side chains of Lys114, Lys125, and Arg129 of antithrombin in heparin binding

The anticoagulant polysaccharide heparin binds and activates the plasma proteinase inhibitor antithrombin through a pentasaccharide sequence. Lys114, Lys125, and Arg129 are the three most important residues of the inhibitor for pentasaccharide binding. To elucidate to what extent another positively charged side chain can fulfill the role of each of these residues, we have mutated Lys114 and Lys125 to Arg and Arg129 to Lys. Lys114 could be reasonably well replaced with Arg with only an approximately 15-fold decrease in pentasaccharide affinity, in contrast to an approximately 10(5)-fold decrease caused by substitution with an noncharged amino acid of comparable size. However, a loss of approximately one ionic interaction on mutation to Arg indicates that the optimal configuration of the network of basic residues of antithrombin that together interact with the pentasaccharide requires a Lys in position 114. Replacement of Lys125 with Arg caused an even smaller, approximately 3-fold, decrease in pentasaccharide affinity, compared with that of approximately 400-fold caused by mutation to a neutral amino acid. An Arg in position 125 is thus essentially equivalent to the wild-type Lys in pentasaccharide binding. Substitution of Arg129 with Lys decreased the pentasaccharide affinity an appreciable approximately 100-fold, a loss approaching that of approximately 400-fold caused by substitution with a neutral amino acid. Arg is thus specifically required in position 129 for high-affinity pentasaccharide binding. This requirement is most likely due to the ability of Arg to interact with other residues of antithrombin, primarily, Glu414 and Thr44, in a manner that appropriately positions the Arg side chain for keeping the pentasaccharide anchored to the activated state of the inhibitor.     

Crystal structures of a type-1 ribosome inactivating protein from Momordica balsamina in the bound and unbound states

The ribosome inactivating proteins (RIPs) of type 1 are plant toxins that eliminate adenine base selectively from the single stranded loop of rRNA. We report six crystal structures, type 1 RIP from Momordica balsamina (A), three in complexed states with ribose (B), guanine (C) and adenine (D) and two structures of MbRIP-1 when crystallized with adenosine triphosphate (ATP) (E) and 2'-deoxyadenosine triphosphate (2'-dATP) (F). These were determined at 1.67?, 1.60?, 2.20?, 1.70?, 2.07? and 1.90? resolutions respectively. The structures contained, (A) unbound protein molecule, (B) one protein molecule and one ribose sugar, (C) one protein molecule and one guanine base, (D) one protein molecule and one adenine base, (E) one protein molecule and one ATP-product adenine molecule and (F) one protein molecule and one 2'-dATP-product adenine molecule. Three distinct conformations of the side chain of Tyr70 were observed with (i) χ(1)=-66°and χ(2)=165° in structures (A) and (B); (ii) χ(1)=-95° and χ(2)=70° in structures (C), (D) and (E); and (iii) χ(1)=-163° and χ(2)=87° in structure (F). The conformation of Tyr70 in (F) corresponds to the structure of a conformational intermediate. This is the first structure which demonstrates that the slow conversion of DNA substrates by RIPs can be trapped during crystallization.     

A plant viral coat protein RNA binding consensus sequence contains a crucial arginine.

A defining feature of alfalfa mosaic virus (AMV) and ilarviruses [type virus: tobacco streak virus (TSV)] is that, in addition to genomic RNAs, viral coat protein is required to establish infection in plants. AMV and TSV coat proteins, which share little primary amino acid sequence identity, are functionally interchangeable in RNA binding and initiation of infection. The lysine-rich amino-terminal RNA binding domain of the AMV coat protein lacks previously identified RNA binding motifs. Here, the AMV coat protein RNA binding domain is shown to contain a single arginine whose specific side chain and position are crucial for RNA binding. In addition, the putative RNA binding domain of two ilarvirus coat proteins, TSV and citrus variegation virus, is identified and also shown to contain a crucial arginine. AMV and ilarvirus coat protein sequence alignment centering on the key arginine revealed a new RNA binding consensus sequence. This consensus may explain in part why heterologous viral RNA-coat protein mixtures are infectious.