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PubMed2003

A novel RNA motif based on the structure of unusually stable 2',5'-linked r(UUCG) loops.

We have recently shown that hairpins containing 2',5'-linked RNA loops exhibit superior thermodynamic stability compared to native hairpins comprised of 3',5'-RNA loops [Hannoush, R. N.; Damha, M. J. J. Am. Chem. Soc. 2001, 123, 12368-12374]. A remarkable feature of the 2',5'-r(UUCG) tetraloop is that, unlike the corresponding 3',5'-linked tetraloop, its stability is virtually independent of the hairpin stem composition. Here, we determine the solution structure of unusually stable hairpins of the sequence 5'-G(1)G(2)A(3)C(4)-(U(5)U(6)C(7)G(8))-G(9)(U/T(10))C(11)C(12)-3' containing a 2',5'-linked RNA (UUCG) loop and either an RNA or a DNA stem. The 2',5'-linked RNA loop adopts a new fold that is completely different from that previously observed for the native 3',5'-linked RNA loop. The 2',5'-RNA loop is stabilized by (a). U5.G8 wobble base pairing, with both nucleotide residues in the anti-conformation, (b). extensive base stacking, and (c). sugar-base and sugar-sugar contacts, all of which contribute to the extra stability of this hairpin structure. The U5:G8 base pair stacks on top of the C4:G9 loop-closing base pair and thus appears as a continuation of the stem. The loop uracil U6 base stacks above U5 base, while the cytosine C7 base protrudes out into the solvent and does not participate in any of the stabilizing interactions. The different sugar pucker and intrinsic bonding interactions within the 2',5'-linked ribonucleotides help explain the unusual stability and conformational properties displayed by 2',5'-RNA tetraloops. These findings are relevant for the design of more effective RNA-based aptamers, ribozymes, and antisense agents and identify the 2',5'-RNA loop as a novel structural motif.

Base Sequence↗
PubMed2002

Key issues in the computational simulation of GPCR function: representation of loop domains.

Some key concerns raised by molecular modeling and computational simulation of functional mechanisms for membrane proteins are discussed and illustrated for members of the family of G protein coupled receptors (GPCRs). Of particular importance are issues related to the modeling and computational treatment of loop regions. These are demonstrated here with results from different levels of computational simulations applied to the structures of rhodopsin and a model of the 5-HT2A serotonin receptor, 5-HT2AR. First, comparative Molecular Dynamics (MD) simulations are reported for rhodopsin in vacuum and embedded in an explicit representation of the membrane and water environment. It is shown that in spite of a partial accounting of solvent screening effects by neutralization of charged side chains, vacuum MD simulations can lead to severe distortions of the loop structures. The primary source of the distortion appears to be formation of artifactual H-bonds, as has been repeatedly observed in vacuum simulations. To address such shortcomings, a recently proposed approach that has been developed for calculating the structure of segments that connect elements of secondary structure with known coordinates, is applied to 5-HT2AR to obtain an initial representation of the loops connecting the transmembrane (TM) helices. The approach consists of a simulated annealing combined with biased scaled collective variables Monte Carlo technique, and is applied to loops connecting the TM segments on both the extra-cellular and the cytoplasmic sides of the receptor. Although this initial calculation treats the loops as independent structural entities, the final structure exhibits a number of interloop interactions that may have functional significance. Finally, it is shown here that in the case where a given loop from two different GPCRs (here rhodopsin and 5-HT2AR) has approximately the same length and some degree of sequence identity, the fold adopted by the loops can be similar. Thus, in such special cases homology modeling might be used to obtain initial structures of these loops. Notably, however, all other loops in these two receptors appear to be very different in sequence and structure, so that their conformations can be found reliably only by ab initio, energy based methods and not by homology modeling.

Algorithms↗
PubMed1998

Split-receptors in the tachykinin neurokinin-1 system--mutational analysis of intracellular loop 3.

Several membrane proteins have been functionally expressed from non-covalently coupled, contiguous segments especially with the split-site located between natural domains. Experiments using such 'split-proteins' were here performed in the tachykinin neurokinin-1 (NK1) receptor with co-expression of contiguous segments with split-sites positioned in various intracellular and extracellular loops. The construct where the split-site was located in intracellular loop 3 gave a reasonable expression level of substance-P-binding sites, i.e. 12% of wild-type expression. Of the other split-receptors tested, only the one with the split-site located just outside transmembrane (TM) segment-V gave any detectable substance P binding, which however only was 1% of the wild-type expression level. The construct with the split-site located in intracellular loop 3 bound all of the tested peptide agonists and non-peptide antagonists with normal affinity and was able to stimulate inositol phosphate turnover with a normal EC50 for substance P and an Emax according to the expression level. When intracellular loop 3 was either extended with 112 amino acid residues derived from the muscarine M2 receptor or, when major parts of the loop were deleted in the non-split NK1 receptor, the affinity for neither substance P nor for the prototype nonpeptide antagonist, CP96,345 was affected, yet an increase in EC50 for substance P was observed. Also in the split-receptor, most of intracellular loop 3 could be substituted or even deleted without affecting ligand affinity, although a decreased expression level was observed in constructs having major deletions. It is concluded, that the NK1 receptor is preferentially reconstituted by co-expression of a putative A-domain including TM-I-V and a B-domain including TM-VI and -VII. It is suggested that a number of rhodopsin-like 7TM receptors may function as two-domain structures based on the finding that a network of short loops has been highly conserved within each of the putative domains and, that these domains are separated by a relatively long and in respect of length poorly conserved loop, i.e. intracellular loop 3.

Amino Acid Sequence↗
PubMed2000

Closed- and open-loop handwriting performance in patients with multiple sclerosis.

Normal subjects use an open-loop motor control strategy in handwriting, but they are able to switch to closed-loop motor control when the demands on accuracy increase. These closed-loop handwriting movements of normal subjects resemble the inefficient movements found in writing-impaired patients. The hypothesis that such movement deficits may in fact reflect the use of a closed-loop strategy was tested in a group of writing-impaired patients with multiple sclerosis (MS). The handwriting movements of 10 MS patients and 20 control subjects were examined with a digitizing tablet. Three conditions were used: a standard writing task (expt 1), a closed-loop condition (expt 2), and an open-loop condition (expt 3). Individual stroke movements were analysed. Stroke duration and segmentation were increased for MS patients in the standard writing task. The same was found for control subjects when they wrote under closed-loop conditions. However, under open-loop conditions, the handwriting movements of the MS patients were as fast and fluent as that of control subjects. The results support the hypothesis that the movement characteristics of the writing-impaired MS patients reflect an inadequate use of a closed-loop motor control strategy.

Adult↗
PubMed2001

Solution structure of the A loop of 23S ribosomal RNA.

The A loop is an essential RNA component of the ribosome peptidyltransferase center that directly interacts with aminoacyl (A)-site tRNA. The A loop is highly conserved and contains a ubiquitous 2'-O-methyl ribose modification at position U2552. Here, we present the solution structure of a modified and unmodified A-loop RNA to define both the A-loop fold and the structural impact of the U2552 modification. Solution data reveal that the A-loop RNA has a compact structure that includes a noncanonical base pair between C2556 and U2552. NMR evidence is presented that the N3 position of C2556 has a shifted pKa and that protonation at C2556-N3 changes the C-U pair geometry. Our data indicate that U2552 methylation modifies the A-loop fold, in particular the dynamics and position of residues C2556 and U2555. We compare our structural data with the structure of the A loop observed in a recent 50S crystal structure [Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. (2000) Science 289, 905--920; Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz, T. A. (2000) Science 289, 920--930]. The solution and crystal structures of the A loop are dramatically different, suggesting that a structural rearrangement of the A loop must occur on docking into the peptidyltransferase center. Possible roles of this docking event, the shifted pKa of C2556 and the U2552 2'-O-methylation in the mechanism of translation, are discussed.

Base Pairing↗
PubMed2006

Toward understanding actin activation of myosin ATPase: the role of myosin surface loops.

To understand the complicated interplay when a traveling myosin head reaches interaction distance with two actins in a filament we looked to three myosin loops that early on exert their influences from the "outside" of the myosin. On these we conduct, functionally test, and interpret strategically chosen mutations at sites thought from crystallography to be a patch for binding the "first" of the two actins. One loop bears a hydrophobic triplet of residues, one is the so-called "loop 2," and the third is the "cardiomyopathy" loop. So far as we know, the myosin sites that first respond are the two lysine-rich loops that produce an ionic strength-dependent weak-binding complex with actin. Subsequently, the three loops of interest bind the first actin simultaneously, and all three assist in closing the cleft in the 50-kDa domain of the myosin, a closure that results in transition from weak to strong binding and precedes rapid Pi release and motility. Mutational analysis shows that each such loop contact is distinctive in the route by which it communicates with its specific target elsewhere in myosin. The strongest contact with actin, for example, is that of the triplet-bearing loop. On the other hand, that of loop 2 (dependent on drawing close two myosin lysines and two actin aspartates) is probably responsible for opening switch I and uncovering the gamma-phosphate moiety of bound ATP. Taking into account these findings, we begin to arrange in order many molecular events in muscle function.

Actins↗
PubMed1996

The role of surface loops (residues 204-216 and 627-646) in the motor function of the myosin head.

A characteristic feature of all myosins is the presence of two sequences which despite considerable variations in length and composition can be aligned with loops 1 (residues 204-216) and 2 (residues 627-646) in the chicken myosin-head heavy chain sequence. Recently, an intriguing hypothesis has been put forth suggesting that diverse performances of myosin motors are achieved through variations in the sequences of loops 1 and 2 [Spudich, J. (1994) Nature (London) 372, 515-518]. Here, we report on the study of the effects of tryptic digestion of these loops on the motor and enzymatic functions of myosin. Tryptic digestions of myosin, which produced heavy meromyosin (HMM) with different percentages of molecules cleaved at both loop 1 and loop 2, resulted in the consistent decrease in the sliding velocity of actin filaments over HMM in the in vitro motility assays, did not affect the Vmax, and increased the Km values for actin-activated ATPase of HMM. Selective cleavage of loop 2 on HMM decreased its affinity for actin but did not change the sliding velocity of actin in the in vitro motility assays. The cleavage of loop 1 and HMM decreased the mean sliding velocity of actin in such assays by almost 50% but did not alter its affinity for HMM. To test for a possible kinetic determinant of the change in motility, 1-N6-ethenoadenosine diphosphate (epsilon-ADP) release from cleaved and uncleaved myosin subfragment 1 (S1) was examined. Tryptic digestion of loop 1 slightly accelerated the release of epsilon-ADP from S1 but did not affect the rate of epsilon-ADP release from acto-S1 complex. Overall, the results of this work support the hypothesis that loop 1 can modulate the motor function of myosin and suggest that such modulation involves a mechanism other than regulation of ADP release from myosin.

Actins↗
PubMed1996

Fibrinogen assembly and secretion. Role of intrachain disulfide loops.

Human fibrinogen is a homodimer composed of three different (Aalpha, Bbeta, gamma) polypeptide chains. The chains are linked by 29 inter- and intrachain disulfide bonds. Each half-molecule has 6 intrachain disulfide bonds, which form loops in the carboxyl-terminal region of each of the chains. Aalpha chain has one disufide loop (Cys442-Cys472), Bbeta has three (Cys201-Cys286, Cys211-Cys240, and Cys394-Cys407), and gamma has two loops (Cys153-Cys182 and Cys326-Cys339). The intrachain loops are conserved in fibrinogens of different species. We changed, by site-directed mutagenesis, the cysteines, which form the intrachain loops, to serine or alanine. Fibrinogen chain assembly and secretion was determined in transiently transfected COS cells expressing two normal and a mutant fibrinogen chain. In the Bbeta and gamma chains, disruption of the disulfide loops closest to the "coiled-coil" region (CysBbeta211-Cys240, CysBbeta201-Cys286, and Cysgamma153-Cys182) abolished chain assembly and secretion, indicating that the disulfide loops closest to the coiled-coil region are essential for chain assembly. By contrast, preventing formation of the disulfide loops, which are toward the carboxyl termini of each of the chains, had different effects. Disruption of the single Aalpha disulfide loop had no effect, as did disruption of BbetaCys394-Cys407. However, disruption of Cysgamma326-Cys339, which is similar in size and location to CysBbeta394-Cys407, allowed chain assembly to occur, but the assembled chains were not secreted.

Animals↗
PubMed1997

Intrinsic specificity of the reactive site loop of alpha1-antitrypsin, alpha1-antichymotrypsin, antithrombin III, and protease nexin I.

Members of the serpin (serine protease inhibitor) family share a similar backbone structure but expose a variable reactive-site loop, which binds to the catalytic groove of the target protease. Specificity originates in part from the sequence of this loop and also from secondary binding sites that contribute to the inhibitor function. To clarify the intrinsic contribution of the reactive-site loop, alpha1-antichymotrypsin has been utilized as a scaffold to construct chimeras carrying the loop of antithrombin III, protease nexin 1, or alpha1-antitrypsin. Reactive-site loops not only vary in sequence but also in length; therefore, the length of the reactive-site loop was also varied in the chimeras. The efficacy of the specificity transfer was evaluated by measuring the stoichiometry of the reaction, the ability to form an SDS-stable complex, and the association rate constant with a number of potential targets (chymotrypsin, neutrophil elastase, trypsin, thrombin, factor Xa, activated protein C, and urokinase). Overall, substitution of a reactive-site loop was not sufficient to transfer the specificity of a given serpin to alpha1-antichymotrypsin. Specificity of the chimera partly matched that of the loop donor and partly that of the acceptor, whereas the behavior as an inhibitor or a substrate depended upon the targeted protease. Results suggest that, aside from the contributions of the loop sequence and the framework-specific secondary binding sites, an intramolecular control may be essential for productive interaction.

Amino Acid Sequence↗
PubMed1997

The role of the cystine loop in acetylcholine receptor assembly.

Nicotinic acetylcholine receptors (AChRs) are composed of alpha, beta, gamma, and delta subunits, assembled into alpha2betagammadelta pentamers. A highly conserved feature of ionotropic neurotransmitter receptors, such as AChRs, is a 15-amino acid cystine "loop." We find that an intact cystine loop is necessary for complete AChR assembly. By preventing formation of the loop with 5 mM dithiothreitol, AChR subunits assemble into alphabetagamma trimers, but the subsequent steps in assembly are blocked. When alpha subunit loop cysteines are mutated to serines, assembly is blocked at the same step as with dithiothreitol. In contrast, when beta subunit loop cysteines are mutated to serines, assembly is blocked at a later step, i.e. after assembly of alphabetagammadelta tetramers and before the addition of the second alpha subunit. After formation of the cystine loop, the alpha subunit undergoes a conformational change, which buries the loop. This conformational change is concurrent with the step in assembly blocked by removal of the disulfide bond of the cystine loop, i.e. after assembly of alphabetagamma trimers and before the addition of the delta subunit. The data indicate that the alpha subunit conformational change involving the cystine loop is key to a series of folding events that allow the addition of unassembled subunits.

Amino Acid Sequence↗
PubMed2002

5' stem-loop of collagen alpha 1(I) mRNA inhibits translation in vitro but is required for triple helical collagen synthesis in vivo.

The 5' stem-loop is a conserved sequence element found around the translation initiation site of three collagen mRNAs, alpha1(I), alpha2(I), and alpha1(III). We show here that the 5' stem-loop of collagen alpha1(I) mRNA is inhibitory to translation in vitro. The sequence 5' to the translation initiation codon, as a part of the 5' stem-loop, is also not efficient in initiating translation under competitive conditions. This suggests that collagen alpha1(I) mRNA may not be a good substrate for translation. Since the 5' stem-loop binds protein factors in collagen-producing cells, this binding may regulate its translation in vivo. We studied in vivo translation of collagen alpha1(I) mRNA after transfecting collagen alpha1(I) genes with and without the 5' stem-loop into Mov 13 fibroblasts. The mRNA with the alpha1(I) 5' stem-loop was translated into pepsin-resistant collagen, which was secreted into the cellular medium. This mRNA also produced more disulfide-bonded high molecular weight collagen found intracellularly. The mRNA in which the 5' stem-loop was mutated, but without affecting the coding region of the gene, was translated into pepsin-sensitive collagen and produced only trace amounts of disulfide-bonded collagen. This suggests that the 5' stem-loop is required for proper folding or stabilization of the collagen triple helix. To our knowledge this is the first example that an RNA element located in the 5'-untranslated region is involved in synthesis of a secreted multisubunit protein. We suggest that 5' stem-loop, with its cognate binding proteins, targets collagen mRNAs for coordinate translation and couples translation apparatus to the rest of the collagen biosynthetic pathway.

5' Untranslated Regions↗
PubMed2003

Functional roles of loops 3 and 4 in the cyclic nucleotide binding domain of cyclic AMP receptor protein from Escherichia coli.

Cyclic AMP is a ubiquitous secondary message that regulates a large variety of functions. The protein structural motif that binds cAMP is highly conserved with the exception of loops 3 and 4, whose structure and length are variable. The cAMP receptor protein of Escherichia coli, CRP, was employed as a model system to elucidate the functional roles of these loops. Based on the sequence differences between CRP and cyclic nucleotide gated channel, three mutants of CRP were constructed: deletion (residues 54-56 in loop 3 were deleted), insertion (loop 4 was lengthened by 5 residues between Glu-78 and Gly-79) and double mutants. The effects of these mutations on the structure and function of CRP were monitored. Results show that the deletion and insertion mutations do not significantly change the secondary structure of CRP, although the tertiary and quaternary structures are perturbed. The functional data indicate that loop 3 modulates the binding affinities of cAMP and DNA. Although the lengthened loop 4 may have some fine-tuning functions, the specific function of the original loop 4 of CRP remains uncertain. The function consequences of mutation in loop 3 of CRP are similar to that of site A and site B in the regulatory subunits of cyclic AMP-dependent protein kinases. Thus, the roles played by loop 3 in CRP may represent a more common mechanism employed by cyclic nucleotide binding domain in modulating ligand binding affinity and intramolecular communication.

Amino Acid Sequence↗
PubMed2004

The myosin cardiac loop participates functionally in the actomyosin interaction.

The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on beta-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in beta-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using site-directed mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.

Actomyosin↗
PubMed2004

Probing the active site loop motif of murine ferrochelatase by random mutagenesis.

Ferrochelatase catalyzes the terminal step of the heme biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. A conserved loop motif was shown to form part of the active site and contact the bound porphyrin by molecular dynamics calculations and structural analysis. We applied a random mutagenesis approach and steady-state kinetic analysis to assess the role of the loop motif in murine ferrochelatase function, particularly with respect to porphyrin interaction. Functional substitutions in the 10 consecutive loop positions Gln(248)-Leu(257) were identified by genetic complementation in Escherichia coli strain Deltavis. Lys(250), Val(251), Pro(253), Val(254), and Pro(255) tolerated a variety of replacements including single substitutions and contained low informational content. Gln(248), Ser(249), Gly(252), Trp(256), and Leu(257) possessed high informational content, since permissible replacements were limited and only observed in multiply substituted mutants. Selected active loop variants exhibited k(cat) values comparable with or higher than that of wild-type murine ferrochelatase. The K(m) values for porphyrin increased, except for the single mutant V251L. Other than a moderate increase observed in the triple mutant S249A/K250Q/V251C, the K(m) values for Fe(2+) were lowered. The k(cat)/K(m) for porphyrin remained largely unchanged, with the exception of a 10-fold reduction in the triple mutant K250M/V251L/W256Y. The k(cat)/K(m) for Fe(2+) was improved. Molecular modeling of these active loop variants indicated that loop mutations resulted in alterations of the active site architecture. However, despite the plasticity of the loop primary structure, the relative spatial positioning of the loop in the active site appeared to be maintained in functional variants, supporting a role for the loop in ferrochelatase function.

Amino Acid Motifs↗
PubMed2004

The long and short flavodoxins: I. The role of the differentiating loop in apoflavodoxin structure and FMN binding.

Flavodoxins are well known one-domain alpha/beta electron-transfer proteins that, according to the presence or absence of a approximately 20-residue loop splitting the fifth beta-strand of the central beta-sheet, have been classified in two groups: long and short-chain flavodoxins, respectively. Although the flavodoxins have been extensively used as models to study electron transfer, ligand binding, protein stability and folding issues, the role of the loop has not been investigated. We have constructed two shortened versions of the long-chain Anabaena flavodoxin in which the split beta-strand has been spliced to remove the original loop. The two variants have been carefully analyzed using various spectroscopic and hydrodynamic criteria, and one of them is clearly well folded, indicating that the long loop is a peripheral element of the structure of long flavodoxins. However, the removal of the loop (which is not in contact with the cofactor in the native structure) markedly decreases the affinity of the apoflavodoxin-FMN complex. This seems related to the fact that, in long flavodoxins, the adjacent tyrosine-bearing FMN binding loop (which is longer and thus more flexible than in short flavodoxins) is stabilized in its competent conformation by interactions with the excised loop. The modest role played by the long loop of long flavodoxins in the structure of these proteins (and in its conformational stability, see Lopez-Llano, J., Maldonado, S., Jain, S., Lostao, A., Godoy-Ruiz, R., Sanchez-Ruiz, Cortijo, M., Fernandez-Recio, J., and Sancho, J. (2004) J. Biol. Chem. 279, 47184-47191) opens the possibility that its conservation in so many species is related to a functional role yet to be discovered. In this respect, we discuss the possibility that the long loop is involved in the recognition of some flavodoxin partners. In addition, we report on a structural feature of flavodoxins that could indicate that the short flavodoxins derive from the long ones.

Anabaena↗
PubMed2006

The mitochondrial intermembrane loop region of rat carnitine palmitoyltransferase 1A is a major determinant of its malonyl-CoA sensitivity.

Carnitine palmitoyltransferase (CPT) 1A adopts a polytopic conformation within the mitochondrial outer membrane, having both the N- and C-terminal segments on the cytosolic aspect of the membrane and a loop region connecting the two transmembrane (TM) segments protruding into the inter membrane space. In this study we demonstrate that the loop exerts major effects on the sensitivity of the enzyme to its inhibitor, malonyl-CoA. Insertion of a 16-residue spacer between the C-terminal part of the loop sequence (i.e. between residues 100 and 101) and TM2 (which is predicted to start at residue 102) increased the sensitivity to malonyl-CoA inhibition of the resultant mutant protein by more than 10-fold. By contrast, the same insertion made between TM1 and the loop had no effects on the kinetic properties of the enzyme, indicating that effects on the catalytic C-terminal segment were specifically induced by loop-TM2 interactions. Enhanced sensitivity was also observed in all mutants in which the native TM2-loop pairing was disrupted either by making chimeras in which the loops and TM2 segments of CPT 1A and CPT 1B were exchanged or by deleting successive 9-residue segments from the loop sequence. The data suggest that the sequence spanning the loop-TM2 boundary determines the disposition of this TM in the membrane so as to alter the conformation of the C-terminal segment and thus affect its interaction with malonyl-CoA.

Amino Acid Sequence↗
PubMed1983

Loop stereochemistry and dynamics in transfer RNA.

The stereochemistry and the dynamics of two loops of yeast tRNA-asp, the thymine loop and the anticodon loop, are compared in the hope of a better understanding of the relationships between loop sequence and loop topology. Both loops are seven residues long and both present sharp turns after the second residue, U33 and psi 55, stabilized by hydrogen bonds between N3-H of the pyrimidine and the phosphates of C36 and A58 and stacking interactions of the pyrimidine ring with the phosphates of U35 and A57, respectively. In the thymine loop, the two purines following C56, A57 and A58, open up to leave space for the intercalation of the first invariant guanine residue of the D-loop, while the two pyrimidine bases, which follow A58, turn away from the stacking pattern of the thymine arm and stack instead with the last base pair of the dihydrouridine arm A15-U48. In the anticodon loop, however, the bases G34 to C38 form an helical stack in continuity with the anticodon stem on the 3'-end. At the same time C36 forms Watson-Crick hydrogen bonds with G34 of a twofold symmetrically related molecule. The anticodon-anticodon base pairing interactions between symmetrically-related molecules are stabilized by stacking with the modified base G37 on both sides of the triplet. Some comparisons are made with the structure of yeast tRNA-phe and some implications about the structure of mitochondrial tRNAs are discussed.

Anticodon↗
PubMed1996

Flexible loop in the structure of S-adenosylmethionine synthetase crystallized in the tetragonal modification.

S-Adenosylmethionine synthetase (MAT, ATP:L-methionine S-adenosyltransferase, E.C.2.5.1.6.) plays a central metabolic role in all organisms. MAT catalyzes the two-step reaction which synthesizes S-adenosylmethionine (AdoMet), pyrophosphate (PPi) and orthophosphate (Pi) from ATP and L-methionine. AdoMet is the primary methyl group donor in biological systems. MAT from Escherichia coli was crystallized in the tetragonal modification with space group P4(3)2(1)2 using the same conditions as previously yielded crystals of the hexagonal system [Takusagawa, et al., (1996), J. Biol. Chem. 171, 136-147], except for the crystallization temperature. The structure has been determined by molecular replacement at 3.2 A resolution. The overall structure of the tetrameric MAT in the tetragonal modification is essentially the same as the structure found in the hexagonal modification. However there are two remarkable differences between the structures of two modifications. One is the contents in the active sites (holoform vs. apo-form), and the other is the conformation of the flexible loop over the active site (open vs. closed). These differences in the crystal structures are caused solely by the difference in crystallization temperatures (26 degrees C vs. 4 degrees C). We have interpreted the structural data obtained from the X-ray analyses in conjunction with the results of the mechanistic and sequencing studies in terms of possible dynamic motion of the flexible loop. When a substrate/product binds in the active site (hexagonal modification), the loop becomes disordered, apparently due to flexibility at the entrance of the active site as if it acts as a "mobile loop" during the catalytic reaction. On the other hand, when the temperature is decreased, the dynamic motion of the flexible loop may be reduced, and the loop residues enter the active site and close its entrance (tetragonal modification). Thus, the active site of the tetragonal modification is empty despite the crystals being grown in mother liquor containing a large concentration of phosphate (100 mM). There is no significant displacement of amino acid residues in the active site between the holo and apo forms, suggesting that the flexible loop plays an important role in determination of the contents in the active site. Since the functionally important amino acid residues in the active site are all conserved throughout various species, the structures of the active sites and the mechanism of the catalysis are probably essentially identical in the enzymes from a wide range of organisms. However, the substrate KM and Vmax values of MATs from various species are distributed over a wide range. The amino acid residues in the flexible loop regions are poorly conserved throughout various species. Therefore, the wide differences in catalysis rates of MATs from various speeches may be due to the differences in the composition of the flexible loop.

Amino Acid Sequence↗