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PubMed2001

Large-scale domain movements and hydration structure changes in the active-site cleft of unligated glutamate dehydrogenase from Thermococcus profundus studied by cryogenic X-ray crystal structure analysis and small-angle X-ray scattering.

Here we describe the large-scale domain movements and hydration structure changes in the active-site cleft of unligated glutamate dehydrogenase. Glutamate dehydrogenase from Thermococcus profundus is composed of six identical subunits of M(r) 46K, each with two distinct domains of roughly equal size separated by a large active-site cleft. The enzyme in the unligated state was crystallized so that one hexamer occupied a crystallographic asymmetric unit, and the crystal structure of the hexamer was solved and refined at a resolution of 2.25 A with a crystallographic R-factor of 0.190. In that structure, the six subunits displayed significant conformational variations with respect to the orientations of the two domains. The variation was most likely explained as a hinge-bending motion caused by small changes in the main chain torsion angle of the residue composing a loop connecting the two domains. Small-angle X-ray scattering profiles both at 293 and 338 K suggested that the apparent molecular size of the hexamer was slightly larger in solution than in the crystalline state. These results led us to the conclusion that (i) the spontaneous domain motion was the property of the enzyme in solution, (ii) the domain motion was trapped in the crystallization process through different modes of crystal contacts, and (iii) the magnitude of the motion in solution was greater than that observed in the crystal structure. The present cryogenic diffraction experiment enabled us to identify 1931 hydration water molecules around the hexamer. The hydration structures around the subunits exhibited significant changes in accord with the degree of the domain movement. In particular, the hydration water molecules in the active-site cleft were rearranged markedly through migrations between specific hydration sites in coupling strongly with the domain movement. We discussed the cooperative dynamics between the domain motion and the hydration structure changes in the active site of the enzyme. The present study provides the first example of a visualized hydration structure varying transiently with the dynamic movements of enzymes and may form a new concept of the dynamics of multidomain enzymes in solution.

Binding Sites↗
PubMed1990

A comparison between the binding modes of a substrate and inhibitor to papain as observed in complex crystal structures.

On the basis of the crystal structures of papain complexed with the substrate analogue benzyloxycarbonyl-L-phenylalanyl-L-alanine chloromethyl-ketone (Drenth, J., Kalk, K.H., and Swen, H.M. (1976) Biochemistry 15, 3731-3738) and with the inhibitor E-64-c, the binding modes were compared at the atomic level to clarify the functional difference between the substrate and inhibitor. Irrespective of the reverse chemical bonding in the peptide bonds, both the molecules are located at the S subsites of papain with similar interactions. However, the inhibitory activity of E-64-c is characterized by the stereochemical function of a carboxyoxirane ring and the tight binding of the isopentylaminoleucyl side chain to the S subsites.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Crystal structure of phosphoserine phosphatase from Methanococcus jannaschii, a hyperthermophile, at 1.8 A resolution.

BACKGROUND: D-Serine is a co-agonist of the N-methyl-D-aspartate subtype of glutamate receptors, a major neurotransmitter receptor family in mammalian nervous systems. D-Serine is converted from L-serine, 90% of which is the product of the enzyme phosphoserine phosphatase (PSP). PSP from M. jannaschii (MJ) shares significant sequence homology with human PSP. PSPs and P-type ATPases are members of the haloacid dehalogenase (HAD)-like hydrolase family, and all members share three conserved sequence motifs. PSP and P-type ATPases utilize a common mechanism that involves Mg(2+)-dependent phosphorylation and autodephosphorylation at an aspartyl side chain in the active site. The strong resemblance in sequence and mechanism implies structural similarity among these enzymes. RESULTS: The PSP crystal structure resembles the NAD(P) binding Rossmann fold with a large insertion of a four-helix-bundle domain and a beta hairpin. Three known conserved sequence motifs are arranged next to each other in space and outline the active site. A phosphate and a magnesium ion are bound to the active site. The active site is within a closed environment between the core alpha/beta domain and the four-helix-bundle domain. CONCLUSIONS: The crystal structure of MJ PSP was determined at 1.8 A resolution. Critical residues were assigned based on the active site structure and ligand binding geometry. The PSP structure is in a closed conformation that may resemble the phosphoserine bound state or the state after autodephosphorylation. Compared to a P-type ATPase (Ca(2+)-ATPase) structure, which is in an open state, this PSP structure appears also to be a good model for the closed conformation of P-type ATPase.

Amino Acid Sequence↗
PubMed1998

A 2.3 A resolution structure of chymosin complexed with a reduced bond inhibitor shows that the active site beta-hairpin flap is rearranged when compared with the native crystal structure.

In the crystal structure of uncomplexed native chymosin, the beta-hairpin at the active site, known as 'the flap', adopts a different conformation from that of other aspartic proteinases. This conformation would prevent the mode of binding of substrates/inhibitors generally found in other aspartic proteinase complexes. We now report the X-ray analysis of chymosin complexed with a reduced bond inhibitor CP-113972 ¿(2R,3S)-isopropyl 3-[(L-prolyl-p-iodo-L-phenylalanyl-S-methyl-cysteinyl)amino-4]-cyclohexy l-2-hydroxybutanoate¿ at 2.3 A resolution in a novel crystal form of spacegroup R32. The structure has been refined by restrained least-squares methods to a final R-factor of 0.19 for a total of 11 988 independent reflections in the resolution range 10 to 2.3 A. The extended beta-strand conformation of the inhibitor allows hydrogen bonds within the active site, while its sidechains make both electrostatic and hydrophobic interactions with residues lining the specificity pockets S4-->S1. The flap closes over the active site cleft in a way that closely resembles that of other previously determined aspartic proteinase inhibitor complexes. We conclude that the usual position and conformation of the flap found in other aspartic proteinases is available to native chymosin. The conformation observed in the native crystal form may result from intermolecular interactions between symmetry-related molecules in the crystal lattice.

Animals↗
PubMed2005

Crystal structure of tetrameric homoisocitrate dehydrogenase from an extreme thermophile, Thermus thermophilus: involvement of hydrophobic dimer-dimer interaction in extremely high thermotolerance.

The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-A resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main- and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related beta-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.

Alcohol Oxidoreductases↗
PubMed1987

Crystal structure of Cd,Zn metallothionein.

The crystal structure of Cd,Zn metallothionein isoform II from rat liver has been determined using the anomalous scattering data from five Cd in the native protein. The structure of a 4Cd cluster was solved by direct methods. A 2.3 A resolution electron density map was calculated by an iterative solvent leveling and map inversion procedure. The structure is folded into two domains. The N-terminal domain (beta) of residues 1-29 enfolds a three-metal cluster of 1 Cd and 2 Zn coordinated by six terminal cysteine thiolate ligands and three bridging cysteine thiolates. The C-terminal domain (alpha) of residues 30-61 enfolds a 4Cd cluster coordinated by six terminal and five bridging cysteine thiolates. All seven metal sites have tetrahedral coordination geometry. The domains are roughly spherical, diameter 15-20 A; there is limited contact between domains. The folding of alpha and beta is topologically similar but with opposite chirality. Redundant, short cysteine-containing sequences have similar roles in cluster formation in both alpha and beta. The Cd1Zn2(cys)9 cluster is homologous with a 12 atom fragment of the Cd4(cys)11 cluster.

Animals↗
PubMed2001

Target-induced conformational adaptation of calmodulin revealed by the crystal structure of a complex with nematode Ca(2+)/calmodulin-dependent kinase kinase peptide.

Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM.

Amino Acid Sequence↗
PubMed1999

Three-centre C-H---O hydrogen bonds in the DNA minor groove: analysis of oligonucleotide crystal structures.

AA.TT and GA.TC dinucleotide steps in B-DNA-type oligomeric crystal structures and in protein-bound DNA fragments (solved using data with resolution <2.6 A) show very small variations in their local dinucleotide geometries. A detailed analysis of these crystal structures reveals that in AA.TT and GA.TC steps the electropositive C2-H2 group of adenine is in very close proximity to the keto O atoms of both the pyrimidine bases in the antiparallel strand of the duplex structure, suggesting the possibility of intra-base pair as well as cross-strand inter-base pair C-H---O hydrogen bonds in the DNA minor groove. The C2--H2---O2 hydrogen bonds in the A.T base pairs could be a natural consequence of Watson-Crick pairing. However, the cross-strand interactions between the bases at the 3'-end of the AA.TT and GA.TC steps obviously arise owing to specific local geometry of these steps, since a majority of the H2---O2 distances in both data sets are considerably shorter than their values in the uniform fibre model (3.3 A) and many are even smaller than the sum of the van der Waals radii. The analysis suggests that in addition to already documented features such as the large propeller twist of A.T base pairs and the hydration of the minor groove, these C2-H---O2 cross-strand interactions may also play a role in the narrowing of the minor groove in A-tract regions of DNA and help explain the high structural rigidity and stability observed for poly(dA).poly(dT).

Base Pairing↗
PubMed1996

Crystal Structures and Luminescence Properties of Platinum(II) Complexes Containing 3,3'-Biisoquinoline.

Three new platinum complexes containing 3,3'-biisoquinoline (i-biq), [Pt(CN)(2)(i-biq)] (1), [PtCl(2)(i-biq)] (2), and [Pt(i-biq)(2)](PF(6))(2) (3), have been synthesized as orange-red, yellow, and colorless crystals, respectively. Their crystal structures and luminescence properties are reported. Crystal data: for 1.0.5H(2)O, PtO(0.5)N(4)C(20)H(13), orthorhombic, Pbcm, a = 13.989(2) Å, b = 18.304(1) Å, c = 6.682(3) Å, V = 1710.9(6) Å(3), Z = 4, and final R = 0.039 (R(w) = 0.033) for 970 independent reflections; for 2.DMF.H(2)O, PtCl(2)O(2)N(3)C(21)H(21), triclinic, P&onemacr;, a = 11.047(1) Å, b = 12.397(3) Å, c = 8.000(2) Å, alpha = 106.56(1) degrees, beta = 100.15(1) degrees, gamma = 76.15(1) degrees, V = 1012.8(3) Å(3), Z = 2, and final R = 0.058 (R(w) = 0.077) for 4219 independent reflections; for 3.2DMF, PtP(2)F(12)O(2)N(6)C(42)H(38), triclinic, P&onemacr;, a = 10.795(2) Å, b = 13.511(2) Å, c = 8.281(1) Å, alpha = 105.22(1) degrees, beta = 112.17(1) degrees, gamma = 85.02(1) degrees, V = 1079.2(3) Å(3), Z = 1, and final R = 0.038 (R(w) = 0.042) for 3606 independent reflections. Square-planar complexes of 1 are stacked in the crystal to form a columnar structure with the Pt-Pt distance of 3.34 Å. The crystal emits strongly, even at room temperature, and the emission spectrum is similar to that for the [Pt(CN)(2)(bpy)] crystal (bpy = 2,2'-bipyridine), which is due to a (3)dpi[dsigma(Pt) --> pi(i-biq)] transition. The single crystal emission spectrum at 77 K is, however, observed as a superposition of broad (3)dpi and sharp (3)pipi(i-biq) emissions. The crystal structure of 2 has a completely different stacking structure from that of 1. The stacking occurs on the i-biq ligands, and the Pt atoms are separated more than 6 Å. The complex exhibits only a structured emission component assigned to the (3)pipi(i-biq) transition in the crystal at 77 K, in agreement with the crystal structure with no Pt-Pt interaction. In the crystal of 3, the [Pt(i-biq)(2)](2+) complexes are stacked but offset, being in close contact between parts of adjacent i-biq ligands. There is no Pt-Pt interaction also in this case. Two i-biq ligands in the complex are distorted to adopt the bowed conformation due to the steric crowding of the alpha-hydrogens on opposite ligands. Nevertheless, 3 provides almost the same (3)pipi emission spectrum as 1 and 2 in dilute glassy solution at 77 K. The (3)pipi emission spectra observed in the crystals of these Pt(II) complexes are red-shifted compared with those in dilute glassy solution. The fact is attributable to the pi-pi intermolecular interactions between the ligands in the crystals. The factors controlling the crystal structures for these complexes are also discussed.

Journal Article↗
PubMed2002

A hybrid Monte Carlo method for crystal structure determination from powder diffraction data.

A hybrid Monte Carlo algorithm for crystal structure determination from powder diffraction data is presented. The algorithm combines the key components of molecular dynamics and Monte Carlo simulations to achieve efficient sampling of phase space, allowing the crystal structure of capsaicin to be determined from powder diffraction data more effectively than by a simulated-annealing approach. The implementation of the algorithm, the choice of the simulation parameters and the performance of the algorithm are discussed.

Journal Article↗
PubMed1997

Crystal structure of 2'-O-Me(CGCGCG)2, an RNA duplex at 1.30 A resolution. Hydration pattern of 2'-O-methylated RNA.

The molecular and crystal structure of 2'-O-Me (CGCGCG)2 has been determined using synchrotron radiation at near-atomic resolution (1.30 A), the highest resolution to date in the RNA field. The crystal structure is a half-turn A-type helix with some helical parameters deviating from canonical A-RNA, such as low base pair rise, elevated helical twist and inclination angles. In CG steps, inter-strand guanines are parallel while cytosines are not parallel. In steps GC this motif is reversed. This type of regularity is not seen in other RNA crystal structures. The structure includes 44 water molecules and two hydrated Mg2+ions one of which lies exactly on the crystallographic 2-fold axis. There are distinct patterns of hydration in the major and the minor grooves. The major groove is stabilised by water clusters consisting of fused five- and six-membered rings. Minor groove contains only a single row of water molecules; each water bridges either two self-parallel cytosines or two self-parallel guanines by a pair of hydrogen bonds. The structure provides the first view of the hydration scheme of 2'-O-methylated RNA duplex.

Base Composition↗
PubMed1994

A 1H-NMR determination of the solution structure of the A-chain of insulin: comparison with the crystal structure and an examination of the role of solvent.

The 1H-NMR chemical shift assignments for the oxidized A-chain of bovine insulin have been determined in aqueous and 30% trifluoroethanol/water solutions. Analysis of the observed medium-range nuclear Overhauser effects indicates that in aqueous solution significant populations of the peptide exist, with a 3(10)-helical conformation over residues 12-17. This region corresponds to helix A (13-20) in the crystal structure of the 2 Zn insulin hexamer. In 30% TFE solution, the NOE data are supportive of a random coil conformation throughout the peptide.

Amino Acid Sequence↗
PubMed2005

On the crystal structure of Cr2N precipitates in high-nitrogen austenitic stainless steel.

The crystal structure of Cr(2)N precipitates in high-nitrogen austenitic stainless steel was investigated by transmission electron microscopy (TEM). Based on the analyses of selected area diffraction (SAD) patterns, the crystal structure of Cr(2)N was confirmed to be trigonal (P31m) and was characterized by three sets of superlattice reflections: (001), ((11/33)0)and ((11/33)1). These could be explained in terms of the epsilon-type occupational ordering of nitrogen. The static concentration waves (SCWs) method was applied to describe the ordered superstructure of Cr(2)N. The occupation probability function (OPF) for describing the distribution of N atoms in the Cr(2)N superstructure was derived based on the superlattice reflections obtained in the SAD patterns and could be expressed as: n(r)=c-1/6eta1cos2piz+4/3eta3cos(2pi/3)(x+y+3z). The crystallographic models for epsilon-type ordering, mainly suggested in the Fe-N system, were discussed in comparison to the present model.

Journal Article↗
PubMed1993

Contribution of buried hydrogen bonds to protein stability. The crystal structures of two barnase mutants.

The crystal structures of two barnase mutants, Tyr78-->Phe and Ser91-->Ala, have been determined to 2.2 A resolution. In both cases, a buried hydroxyl group that makes two hydrogen bonds within the protein was replaced by a hydrogen atom. It is found that neither mutation causes any structural changes, within the limits of error, compared with wild-type and so are confirmed to be non-disruptive. Solvent molecules are not observed in the cavities created by removal of the respective hydroxyl groups and no new interactions are introduced. The local water structure surrounding both sites of mutation is well conserved and resembles that of the wild-type. All four water molecules making contacts with the side-chain of residue 78 and two water molecules nearest to residue 91 in the wild-type are found within a sphere of 0.5 A radius, at the equivalent positions of the respective mutant. No new water molecules are found bound to any of the hydrogen bond donor or acceptor residues involved in these two mutation sites. Previous protein engineering experiments established that the solvent-inaccessible phenolic OH of Tyr78 that makes hydrogen bonds with two uncharged groups (main-chain NH and CO) contributes 1.4 kcal mol-1 to protein stability, while the solvent-inaccessible OH of Ser91 that makes hydrogen bonds with an uncharged main-chain NH and a charged group (O gamma 1) contributes 1.9 kcal mol-1. These stability measurements can now be attributed primarily to the loss of the hydrogen bonding interactions because both mutations neither disrupt the respective protein and local solvent structures, upset the overall hydrogen bonding pattern nor introduce new interactions. The mutations Tyr78-->Phe and Ser91-->Ala are thus good examples of "non-disruptive deletions" and the results of mutagenesis can be analysed at the simplest level.

Bacillus↗
PubMed1993

The tetrasaccharide nystose trihydrate: crystal structure analysis and hydrogen bonding.

The crystal structure of nystose trihydrate, O-beta-D-fructofuranosyl-(2-->1)-O-beta-D-fructofuranosyl-(2-->1)-beta-D -fructofuranosyl alpha-D-glucopyranoside trihydrate, C24H42O21.3H2O, has been determined using Cu K alpha X-ray data at 121 K. The space group is P2(1)2(1)2(1), with 4 molecules in a unit cell of dimensions a = 10.155(1), b = 13.506(1), and c = 23.278(2) A. The structure was refined to R = 0.052 and RW = 0.048 for 2734 observed structure amplitudes. The alpha-D-glucopyranose unit of the molecule has the normal 4C1 chair conformation and the three fructofuranose units have twist conformations lying between E3 and 4T5. One of the three water molecules is distributed over two sites: W-3 with occupancy 0.80 and W-3' with occupancy 0.20. All the hydrogen atoms were located on the difference synthesis with the exception of those attached to the low-occupancy water site. All hydroxyls, two of the three linkage oxygen atoms, and the water molecules are involved in a complex three-dimensional network which can be decomposed into a series of infinite chains intersecting at the water molecules to form homo- and hetero-dromic cycles.

Carbohydrate Conformation↗
PubMed1995

Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis.

Crystal structures of the DNA repair enzyme human uracil-DNA glycosylase (UDG), combined with mutational analysis, reveal the structural basis for the specificity of the enzyme. Within the classic alpha/beta fold of UDG, sequence-conserved residues form a positively charged, active-site groove the width of duplex DNA, at the C-terminal edge of the central four-stranded parallel beta sheet. In the UDG-6-aminouracil complex, uracil binds at the base of the groove within a rigid preformed pocket that confers selectivity for uracil over other bases by shape complementary and by main chain and Asn-204 side chain hydrogen bonds. Main chain nitrogen atoms are positioned to stabilize the oxyanion intermediate generated by His-268 acting via nucleophilic attack or general base mechanisms. Specific binding of uracil flipped out from a DNA duplex provides a structural mechanism for damaged base recognition.

Amino Acid Sequence↗
PubMed1995

Crystal structure of the PvuII restriction endonuclease.

Crystal structures have now been determined for the R.PvuII restriction endonuclease as a protein-DNA complex [Cheng et al., EMBO J. 13 (1994) 3927-3935; this report] and in apo-form [Athanasiadis et al., Nature Struc. Biol. 1 (1994) 469-475; our unpublished result]. The structures indicate how the interaction with DNA might proceed [Riddihough, Nature 370 (1994) 78].

Binding Sites↗
PubMed2004

Origins of protein stability revealed by comparing crystal structures of TATA binding proteins.

The crystal structure of TATA binding protein (TBP) from a mesothermophilic archaeon, Sulfolobus acidocaldarius, has been determined at a resolution of 2.0 A with an R factor of 20.9%. By comparing this structure with the structures of TBPs from a hyperthermophilic archaeon and mesophilic eukaryotes, as well as by comparing amino acid sequences of TBPs from archaea, covering a wide range of optimum growth temperatures, two significant determinants of the stability of TBP have been identified: increasing the interior hydrophobicity by interaction between three residues, Val, Leu, and Ile, with further differentiation of the surface, and increasing its hydrophilicity and raising the cost of unfolding. These findings suggest directions along which the stability of TBP can be engineered.

Amino Acid Sequence↗