The crystal structure of the ttCsaA protein: an export-related chaperone from Thermus thermophilus.

The CsaA protein was first characterized in Bacillus subtilis as a molecular chaperone with export-related activities. Here we report the 2.0 Angstrom-resolution crystal structure of the Thermus thermophilus CsaA protein, designated ttCsaA. Atomic structure and experiments in solution revealed a homodimer as the functional unit. The structure of the ttCsaA monomer is reminiscent of the well known oligonucleotide-binding fold, with the addition of extensions at the N- and C-termini that form an extensive dimer interface. The two identical, large, hydrophobic cavities on the protein surface are likely to constitute the substrate binding sites. The CsaA proteins share essential sequence similarity with the tRNA-binding protein Trbp111. Structure-based sequence analysis suggests a close structural resemblance between these proteins, which may extend to the architecture of the binding sites at the atomic level. These results raise the intriguing possibility that CsaA proteins possess a second, tRNA-binding activity in addition to their export-related function.

Structure of the EMAPII domain of human aminoacyl-tRNA synthetase complex reveals evolutionary dimer mimicry.

The EMAPII (endothelial monocyte-activating polypeptide II) domain is a tRNA-binding domain associated with several aminoacyl-tRNA synthetases, which becomes an independent domain with inflammatory cytokine activity upon apoptotic cleavage from the p43 component of the multisynthetase complex. It comprises a domain that is highly homologous to bacterial tRNA-binding proteins (Trbp), followed by an extra domain without homology to known proteins. Trbps, which may represent ancient tRNA chaperones, form dimers and bind one tRNA per dimer. In contrast, EMAPII domains are monomers. Here we report the crystal structure at 1.14 Angstroms of human EMAPII. The structure reveals that the Trbp-like domain, which forms an oligonucleotide-binding (OB) fold, is related by degenerate 2-fold symmetry to the extra-domain. The pseudo-axis coincides with the dyad axis of bacterial TtCsaA, a Trbp whose structure was solved recently. The interdomain interface in EMAPII mimics the intersubunit interface in TtCsaA, and may thus generate a novel OB-fold-based tRNA-binding site. The low sequence homology between the extra domain of EMAPII and either its own OB fold or that of Trbps suggests that dimer mimicry originated from convergent evolution rather than gene duplication.

Structural basis for anticodon recognition by discriminating glutamyl-tRNA synthetase.

Glutamyl-tRNA synthetases (GluRSs) are divided into two distinct types, with regard to the presence or absence of glutaminyl-tRNA synthetase (GlnRS) in the genetic translation systems. In the original 19-synthetase systems lacking GlnRS, the 'non-discriminating' GluRS glutamylates both tRNAGlu and tRNAGln. In contrast, in the evolved 20-synthetase systems with GlnRS, the 'discriminating' GluRS aminoacylates only tRNAGlu. Here we report the 2.4 A resolution crystal structure of a 'discriminating' GluRS.tRNAGlu complex from Thermus thermophilus. The GluRS recognizes the tRNAGlu anticodon bases via two alpha-helical domains, maintaining the base stacking. We show that the discrimination between the Glu and Gln anticodons (34YUC36 and 34YUG36, respectively) is achieved by a single arginine residue (Arg 358). The mutation of Arg 358 to Gln resulted in a GluRS that does not discriminate between the Glu and Gln anticodons. This change mimics the reverse course of GluRS evolution from anticodon 'non-dicsriminating' to 'discriminating'.

Crucial role of the high-loop lysine for the catalytic activity of arginyl-tRNA synthetase.

The presence of two short signature sequence motifs (His-Ile-Gly-His (HIGH) and Lys-Met-Ser-Lys (KMSK)) is a characteristic of the class I aminoacyl-tRNA synthetases. These motifs constitute a portion of the catalytic site in three dimensions and play an important role in catalysis. In particular, the second lysine of the KMSK motif (K2) is the crucial catalytic residue for stabilization of the transition state of the amino acid activation reaction (aminoacyl-adenylate formation). Arginyl-tRNA synthetase (ArgRS) is unique among all of the class I enyzmes, as the majority of ArgRS species lack canonical KMSK sequences. Thus, the mechanism by which this group of ArgRSs achieves the catalytic reaction is not well understood. Using three-dimensional modeling in combination with sequence analysis and site-directed mutagenesis, we found a conserved lysine in the KMSK-lacking ArgRSs upstream of the HIGH sequence motif, which is likely to be a functional counterpart of the canonical class I K2 lysine. The results suggest a plausible partition of the ArgRSs into two major groups, on the basis of the conservation of the HIGH lysine.

Structural basis for double-sieve discrimination of L-valine from L-isoleucine and L-threonine by the complex of tRNA(Val) and valyl-tRNA synthetase.

Valyl-tRNA synthetase (ValRS) strictly discriminates the cognate L-valine from the larger L-isoleucine and the isosteric L-threonine by the tRNA-dependent "double sieve" mechanism. In this study, we determined the 2.9 A crystal structure of a complex of Thermus thermophilus ValRS, tRNA(Val), and an analog of the Val-adenylate intermediate. The analog is bound in a pocket, where Pro(41) allows accommodation of the Val and Thr moieties but precludes the Ile moiety (the first sieve), on the aminoacylation domain. The editing domain, which hydrolyzes incorrectly synthesized Thr-tRNA(Val), is bound to the 3' adenosine of tRNA(Val). A contiguous pocket was found to accommodate the Thr moiety, but not the Val moiety (the second sieve). Furthermore, another Thr binding pocket for Thr-adenylate hydrolysis was suggested on the editing domain.

Crystal structures of phenylalanyl-tRNA synthetase complexed with phenylalanine and a phenylalanyl-adenylate analogue.

The crystal structures of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) complexed with phenylalanine and phenylalaninyl-adenylate (PheOH-AMP), the synthetic analogue of phenylalanyl-adenylate, have been determined at 2.7A and 2.5A resolution, respectively. Both Phe and PheOH-AMP are engulfed in the active site cleft of the catalytic alpha-subunit of PheRS, and neither makes contact with the PheRS beta-subunit. The conformations and binding of Phe are almost identical in both complexes. The recognition of Phe by PheRS is achieved through a mixture of multiple van der Waals interactions and hydrogen bonds. The side-chain of the Phe substrate is sandwiched between the hydrophobic side-chains of Phealpha258 and Phealpha260 on one side, and the main-chain atoms of the two adjacent beta-strands on the other. The side-chains of Valalpha261 and Alaalpha314 form the back wall of the amino acid binding pocket. In addition, PheRS residues (Trpalpha149, Seralpha180, Hisalpha178, Argalpha204, Glnalpha218, and Glualpha220) form a total of seven hydrogen bonds with the main-chain atoms of Phe. The conformation of PheOH-AMP and the network of interactions of its AMP moiety with PheRS are reminiscent of the other class II synthetases. The structural similarity between PheRS and histidyl-tRNA synthetase extends to the amino acid binding site, which is normally unique for each enzyme. The complex structures suggest that the PheRS beta-subunit may affect the first step of the reaction (formation of phenylalanyl-adenylate) through the metal-mediated conserved alpha/beta-subunit interface. The modeling of tyrosine in the active site of PheRS revealed no apparent close contacts between tyrosine and the PheRS residues. This result implies that the proofreading mechanism against activated tyrosine, rather than direct recognition, may play the major role in the PheRS specificity.

Crystallization and preliminary X-ray analysis of the periplasmic receptor (PotF) of the putrescine transport system in Escherichia coli.

The primary receptor (PotF) of the putrescine transport system in E. coli has been crystallized by the hanging-drop vapor-diffusion technique. The crystals belong to the space group P21212 with unit-cell dimensions a = 269.4, b = 82.33 and c = 93.74 A. The crystals diffract beyond 2.2 A with a rotating-anode X-ray source. A complete data set from the native crystals has been collected and processed at 2.3 A resolution. Two heavy-atom derivatives have been prepared from the same Pt compound at 293 and 277 K. The difference Patterson maps revealed completely different major heavy-atom sites between these two derivatives.

A structural basis for transition-state stabilization in antibody-catalyzed hydrolysis: crystal structures of an abzyme at 1. 8 A resolution.

The three-dimensional structure of a catalytic antibody, 6D9, has been solved as a complex with a transition state analog. The structure was determined from two different crystal forms, and was refined at a resolution of 1.8 A. The antibody 6D9, which was induced by immunization with the phosphonate transition state analog 3, hydrolyzes a prodrug of chloramphenicol monoester 1 to generate the parent drug 2. The kinetic studies have shown that the antibody is catalytic by virtue of the theoretical relationship between the affinity for the transition state and the catalytic efficiency (kcat/kuncat=KS/KTSA). The crystal structure makes it possible to visualize the theoretical relationship. A side-chain (Nepsilon) of HisL27D is placed in a key position to make a hydrogen bond to the phosphonate oxygen of the transition state analog with a distance of 2.72 A, suggesting a hydrogen bond to the oxyanion developing in the transition state of the hydrolysis. There are no catalytic residues, other than the histidine, around the phosphonate moiety. In addition, in the antibody-hapten complex, the hapten bears a folded conformation and the two stacked aromatic rings are buried deep in the antigen-combining site through aromatic-aromatic interaction with TrpH100I and TyrH58. The conformation of the bound hapten suggests that the antibody binds the substrate to change the conformation of the ester moiety to a thermodynamically unstable E-form, thereby making it easy for the substrate to reach the transition-state during catalysis. These observations reveal that the catalytic mechanism is explained purely on the basis of the stabilization of the transition state. The refined high resolution structures reported here are envisaged to have an impact on the understanding of other hydrolytic antibodies, since their haptens share some unique features with the hapten used in this study.

Crystal structure and mutational analysis of the Escherichia coli putrescine receptor. Structural basis for substrate specificity.

PotF protein is a periplasmic substrate-binding protein of the putrescine transport system in Escherichia coli. We have determined the crystal structure of PotF protein in complex with the substrate at 2.3-A resolution. The PotF molecule has dimensions of 54 x 42 x 30 A and consists of two similar globular domains. The PotF structure is reminiscent of other periplasmic receptors with a highest structural homology to another polyamine-binding protein, PotD. Putrescine is tightly bound in the deep cleft between the two domains of PotF through 12 hydrogen bonds and 36 van der Waals interactions. The comparison of the PotF structure with that of PotD provides the insight into the differences in the specificity between the two proteins. The PotF structure, in combination with the mutational analysis, revealed the residues crucial for putrescine binding (Trp-37, Ser-85, Glu-185, Trp-244, Asp-247, and Asp-278) and the importance of water molecules for putrescine recognition.

Crystal structure of troponin C in complex with troponin I fragment at 2.3-A resolution.

Troponin (Tn), the complex of three subunits (TnC, TnI, and TnT), plays a key role in Ca2+-dependent regulation of muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we have determined the crystal structure of TnC (two Ca2+ bound state) in complex with the N-terminal fragment of TnI (TnI1-47). The structure was solved by the single isomorphous replacement method in combination with multiple wavelength anomalous dispersion data. The refinement converged to a crystallographic R factor of 22.2% (Rfree = 32.6%). The central, connecting alpha-helix observed in the structure of uncomplexed TnC (TnCfree) is unwound at the center (residues Ala-87, Lys-88, Gly-89, Lys-90, and Ser-91) and bent by 90 degrees. As a result, TnC in the complex has a compact globular shape with direct interactions between the N- and C-terminal lobes, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TnC. The 31-residue long TnI1-47 alpha-helix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC lobes. The amphiphilic C-end of the TnI1-47 alpha-helix is bound in the hydrophobic pocket of the TnC C-lobe through 38 van der Waals interactions. The results indicate the major difference between Ca2+ receptors integrated with the other proteins (TnC in Tn) and isolated in the cytosol (calmodulin). The TnC/TnI1-47 structure implies a mechanism of how Tn regulates the muscle contraction and suggests a unique alpha-helical regulatory TnI segment, which binds to the N-lobe of TnC in its Ca2+ bound conformation.

Enzyme structure with two catalytic sites for double-sieve selection of substrate.

High-fidelity transfers of genetic information in the central dogma can be achieved by a reaction called editing. The crystal structure of an enzyme with editing activity in translation is presented here at 2.5 angstroms resolution. The enzyme, isoleucyl-transfer RNA synthetase, activates not only the cognate substrate L-isoleucine but also the minimally distinct L-valine in the first, aminoacylation step. Then, in a second, "editing" step, the synthetase itself rapidly hydrolyzes only the valylated products. For this two-step substrate selection, a "double-sieve" mechanism has already been proposed. The present crystal structures of the synthetase in complexes with L-isoleucine and L-valine demonstrate that the first sieve is on the aminoacylation domain containing the Rossmann fold, whereas the second, editing sieve exists on a globular beta-barrel domain that protrudes from the aminoacylation domain.

The crystal structure of troponin C in complex with N-terminal fragment of troponin I. The mechanism of how the inhibitory action of troponin I is released by Ca(2+)-binding to troponin C.

Troponin (Tn), the complex of three subunits (TnC, TnI, and TnT), plays a key role in Ca2+ dependent regulation of muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we have determined the crystal structure of TnC in complex with the N-terminal fragment of TnI (TnI1-47). The structure was solved by single isomorphous replacement method in combination with multiple wavelength anomalous dispersion data. The refinement converged to a crystallographic R-factor of 22.2% (R-free = 32.6%). The central, connecting alpha-helix observed in the structure of uncomplexed TnC (TnCfree) is unwound at the center and bent by 90 degrees. As a result, the TnC in the complex has a compact globular shape with direct interactions between the N- and C-lobes, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TnC. The 31-residue long TnI1-47 alpha-helix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC lobes. The amphiphilic C-terminal end of the TnI1-47 alpha-helix is tightly bound in the hydrophobic pocket of the TnC C-lobe through 38 van der Waals interactions. The results indicate the major difference between integrated (TnC) and isolated (calmodulin) Ca2+ receptors. The TnC/TnI1-47 structure suggests the model for a novel regulatory TnI segment bound to TnC and implies the mechanism of how Tn regulates the muscle contraction.

Surface of bacteriorhodopsin revealed by high-resolution electron crystallography.

Bacteriorhodopsin is a transmembrane protein that uses light energy, absorbed by its chromophore retinal, to pump protons from the cytoplasm of bacteria such as Halobacterium salinarium into the extracellular space. It is made up of seven alpha-helices, and in the bacterium forms natural, two-dimensional crystals called purple membranes. We have analysed these crystals by electron cryo-microscopy to obtain images of bacteriorhodopsin at 3.0 A resolution. The structure covers nearly all 248 amino acids, including loops outside the membrane, and reveals the distribution of charged residues on both sides of the membrane surface. In addition, analysis of the electron-potential map produced by this method allows the determination of the charge status of these residues. On the extracellular side, four glutamate residues surround the entrance to the proton channel, whereas on the cytoplasmic side, four aspartic acids occur in a plane at the boundary of the hydrophobic-hydrophilic interface. The negative charges produced by these aspartate residues is encircled by areas of positive charge that may facilitate accumulation and lateral movement of protons on this surface.

DNA-repair enzymes.

Recent crystallographic studies of DNA-repair enzymes have provided the structural basis for the recognition of damaged DNA. The results imply that flipping out of the base is a common and crucial event in DNA repair. Two classes of repair enzymes that recognize distinct types of damage may exist. DNA-repair enzymes that share similar folds and DNA binding motifs have been proposed to belong to a superfamily.

Precluding uracil from DNA.

Two enzymes, dUTP pyrophosphatase and uracil-DNA glycosylase, prevent the misincorporation of uracil into the genome in distinct manners. The atomic structures of these proteins complexed with substrate analogs reveal the structural basis for uracil recognition and suggest a novel mechanism of DNA repair.

The 1.8-A X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding.

The PotD protein from Escherichia coli is one of the components of the polyamine transport system present in the periplasm. This component specifically binds either spermidine or putrescine. The crystal structure of the E. coli PotD protein complexed with spermidine was solved at 1.8 A resolution and revealed the detailed substrate-binding mechanism. The structure provided the detailed conformation of the bound spermidine. Furthermore, a water molecule was clearly identified in the binding site lying between the amino-terminal domain and carboxyl-terminal domain. Through this water molecule, the bound spermidine molecule forms two hydrogen bonds with Thr 35 and Ser 211. Another periplasmic component of polyamine transport, the PotF protein, exhibits 35% sequence identity with the PotD protein, and it binds only putrescine, not spermidine. To understand these different substrate specificities, model building of the PotF protein was performed on the basis of the PotD crystal structure. The hypothetical structure suggests that the side chain of Lys 349 in PotF inhibits spermidine binding because of the repulsive forces between its positive charge and spermidine. On the other hand, putrescine could be accommodated into the binding site without any steric hindrance because its molecular size is much smaller than that of spermidine, and the positively charged amino group is relatively distant from Lys 349.

Crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli.

PotD protein is a periplasmic binding protein and the primary receptor of the polyamine transport system, which regulates the polyamine content in Escherichia coli. The crystal structure of PotD in complex with spermidine has been solved at 2.5-A resolution. The PotD protein consists of two domains with an alternating beta-alpha-beta topology. The polyamine binding site is in a central cleft lying in the interface between the domains. In the cleft, four acidic residues recognize the three positively charged nitrogen atoms of spermidine, while five aromatic side chains anchor the methylene backbone by van der Waals interactions. The overall fold of PotD is similar to that of other periplasmic binding proteins, and in particular to the maltodextrin-binding protein from E. coli, despite the fact that sequence identity is as low as 20%. The comparison of the PotD structure with the two maltodextrin-binding protein structures, determined in the presence and absence of the substrate, suggests that spermidine binding rearranges the relative orientation of the PotD domains to create a more compact structure.

Atomic model of a pyrimidine dimer excision repair enzyme complexed with a DNA substrate: structural basis for damaged DNA recognition.

T4 endonuclease V is a DNA repair enzyme from bacteriophage T4 that catalyzes the first reaction step of the pyrimidine dimer-specific base excision repair pathway. The crystal structure of this enzyme complexed with a duplex DNA substrate, containing a thymine dimer, has been determined at 2.75 A resolution. The atomic structure of the complex reveals the unique conformation of the DNA duplex, which exhibits a sharp kink with a 60 degree inclination at the central thymine dimer. The adenine base complementary to the 5' side of the thymine dimer is completely flipped out of the DNA duplex and trapped in a cavity on the protein surface. These structural features allow an understanding of the catalytic mechanism and implicate a general mechanism of how other repair enzymes recognize damaged DNA duplexes.

Crystal structure of a pyrimidine dimer-specific excision repair enzyme from bacteriophage T4: refinement at 1.45 A and X-ray analysis of the three active site mutants.

Crystallographic study of bacteriophage T4 endonuclease V, which is involved in the initial step of the pyrimidine dimer-specific excision repair pathway, has been carried out with respect to the wild-type and three different mutant enzymes. This enzyme catalyzes the cleavage of the N-glycosyl bond at the 5'-side of the pyrimidine dimer, and subsequently incises the phosphodiester bond at the apyrimidinic site through a beta-elimination reaction. The structure of the wild-type enzyme refined at 1.45 A resolution reveals the detailed molecular architecture. The enzyme is composed of a single compact domain classified as an all-alpha structure. The molecule is stabilized mainly by three hydrophobic cores, two of which include many aromatic side-chain interactions. The structure has a unique folding motif, where the amino-terminal segment penetrates between two major alpha-helices and prevents their direct contact, and it is incompatible with the close-packing category of helices for protein folding. The concave surface, covered with many positive charges, implies an interface for DNA binding. The glycosylase catalytic center, which comprises Glu23 and the surrounding basic residues Arg3, Arg22 and Arg26, lie in this basic surface. The crystal structures of the three active-site mutants, in which Glu23 was replaced by Gln(E23Q) and Asp (E23D), respectively, and Arg3 by Gln (R3Q), have been determined at atomic resolution. The backbone structures of the E23Q and R3Q mutants were almost identical with that of the wild-type, while the E23D mutation induces a small, but significant, change in the backbone structure, such as an increase of the central kink of the H1 helix at Pro25. In the catalytic center of the glycosylase, however, these three mutations do not generate notable movements of protein atoms, except for significant shifts of some bound water molecules. Thus, the structural differences between the wild-type and each mutant are confined to the remarkably small region around their replaced chemical groups. Combined with the biochemical studies and the difference circular dichroism measurements, these results allow us to conclude that the negatively charged carboxyl group of Glu23 is essential for the cleavage of the N-glycosyl bond, and that the positively charged guanidino group of Arg3 is crucial to bind the substrate, a DNA duplex containing a pyrimidine dimer. The amino terminal alpha-amino group is located at a position approximately 4.4 A away from the carboxyl group of Glu23. These structural features are generally consistent with the reaction scheme proposed by Dodson and co-workers.