Full-matrix least-squares refinement of lysozymes and analysis of anisotropic thermal motion

Crystal structures of turkey egg lysozyme (TEL) and human lysozyme (HL) were refined by full-matrix least-squares method using anisotropic temperature factors. The refinement converged at the conventional R-values of 0.104 (TEL) and 0.115 (HL) for reflections with F_o > 0 to the resolution of 1.12 Å and 1.15 Å, respectively. The estimated r.m.s. coordinate errors for protein atoms were 0.031 Å (TEL) and 0.034 Å (HL). The introduction of anisotropic temperature factors markedly reduced the R-value but did not significantly affect the main chain coordinates. The degree of anisotropy of atomic thermal motion has strong positive correlation with the square of distance from the molecular centroid. The ratio of the radial component of thermal ellipsoid to the r.m.s. magnitude of three principal components has negative correlation with the distance from the molecular centroid, suggesting the domination of libration rather than breathing motion. The TLS model was applied to elucidate the characteristics of the rigid-body motion. The TLS tensors were determined by the least-squares fit to observed temperature factors. The profile of the magnitude of reproduced temperature factors by the TLS method well fitted to that of observed B_eqv. However, considerable disagreement was observed in the shape and orientation of thermal ellipsoid for atoms with large temperature factors, indicating the large contribution of local motion. The upper estimate of the external motion, 67% (TEL) and 61% (HL) of B_eqv, was deduced from the plot of the magnitude of TLS tensors determined for main chain atoms which were grouped into shells according to the distance from the center of libration. In the external motion, the translational portion is predominant and the contribution of libration and screw motion is relatively small. The internal motion, estimated by subtracting the upper estimate of the external motion from the observed temperature factor, is very similar between TEL and HL in spite of the difference in 54 of 130 amino acid residues and in crystal packing, being suggested to reflect the intrinsic internal motion of chicken-type lysozymes. Proteins 30:232–243, 1998. © 1998 Wiley-Liss, Inc.     

Atomic resolution structure of prokaryotic phospholipase A2: analysis of internal motion and implication for a catalytic mechanism

We have found a secreted phospholipase A(2) (PLA(2), EC 3.1.1.4) from Streptomyces violaceoruber A-2688, which is the first PLA(2) identified in prokaryote, and determined its tertiary structure by NMR and X-ray analyses. In this study, we collected the X-ray diffraction data of the bacterial PLA(2) at room temperature (297 K) using conventional MoK(alpha) radiation and refined the structure at a 1.05 A resolution. The atomic resolution analysis led us to introduce disordered conformations and hydrogen atoms into a full anisotropic model. The molecular motion, which is expressed as the sum of rigid-body motion and internal motion of protein, is roughly estimated as the thermal motion when the X-ray diffraction data are collected at room temperature. In this study, we applied a TLS (rigid-body motion in terms of translation, libration, and screw motions) model to analyze the rigid-body motion of the bacterial PLA(2) and calculated the internal motion by subtracting the estimate of the rigid-body motion from the observed anisotropic temperature factor. We also subjected the TLS model to estimate the internal motion of the bovine pancreatic PLA(2) using the anisotropic temperature factor deposited in the Protein Data Bank. Both results indicate that the localization of regions exhibiting larger internal motion in the bacterial PLA(2) is almost the same as that in the bovine pancreatic PLA(2), suggesting that although the tertiary structure of the bacterial PLA(2) is strikingly different from that of the bovine pancreatic PLA(2), the internal motion, which is associated with the calcium(II) ion-binding, phospholipid-binding, and allosteric interfacial activation, is commonly observed in both PLA(2)s.     

Crystallographic analysis of the thermal motion of the inclusion complex of cyclomaltoheptaose (beta-cyclodextrin) with hexamethylenetetramine

The crystal structure of the inclusion complex of cyclomaltoheptaose (beta-cyclodextrin) with hexamethylenetetramine was determined at temperatures of 123, 173, 223, and 293 K. The rigid-body motion of the host and guest molecules was evaluated by means of the TLS method that represents the molecular motion in terms of translation, libration, and screw motion. In increasing the temperature from 123 to 293 K, the amplitude of the rigid body vibration of the host molecule was increased from 1.0 to 1.3 degrees in the rotational motion and from 0.16 to 0.17 A in the translational motion. The cyclomaltoheptaose molecule has the flexibility in seven alpha-(1-->4)-linkages, and each glucose unit was in the rotational vibration around an axis through two glycosidic oxygen atoms. As a result, the rigid-body parameters of cyclomaltoheptaose were considered to be overestimated because of including the contribution from the local motion of glucose units. In contrast, for the guest molecule having no structural flexibility, the TLS analysis demonstrated that the atomic thermal vibration was mostly derived from the rigid body motion. The rotational amplitude of hexamethylenetetramine was changed from 5.2 to 6.6 degrees in increasing the temperature from 123 to 293 K, while the change of the translational amplitude was from 0.20 to 0.23 A. The translational motion of the guest molecule was hindered by the inside wall of the host cavity. The molecular motion was characterized by the rotational vibration around the axis through two nitrogen atoms that were involved in the hydrogen-bond formation.     

Structural origin of weakly ordered nitroxide motion in spin‐labeled proteins

A disulfide-linked nitroxide side chain (R1) used in site-directed spin labeling of proteins often exhibits an EPR spectrum characteristic of a weakly ordered z-axis anisotropic motion at topographically diverse surface sites, including those on helices, loops and edge strands of β-sheets. To elucidate the origin of this motion, the first crystal structures of R1 that display simple z-axis anisotropic motion at solvent-exposed helical sites (131 and 151) and a loop site (82) in T4 lysozyme have been determined. Structures of 131R1 and 151R1 determined at cryogenic or ambient temperature reveal an intraresidue C_α—H···S_δ interaction that immobilizes the disulfide group, consistent with a model in which the internal motions of R1 are dominated by rotations about the two terminal bonds (Columbus, Kálai, Jeko, Hideg, and Hubbell, Biochemistry 2001;40:3828–3846). Remarkably, the 131R1 side chain populates two rotamers equally, but the EPR spectrum reflects a single dominant dynamic population, showing that the two rotamers have similar internal motion determined by the common disulfide-backbone interaction. The anisotropic motion for loop residue 82R1 is also accounted for by a common disulfide-backbone interaction, showing that the interaction does not require a specific secondary structure. If the above observations prove to be general, then significant variations in order and rate for R1 at noninteracting solvent-exposed helical and loop sites can be assigned to backbone motion because the internal motion is essentially constant.     

Crystallographic and functional studies of a modified form of eosinophil-derived neurotoxin (EDN) with novel biological activities

The crystal structure of a post-translationally modified form of eosinophil-derived neurotoxin (EDN) with four extra residues on its N terminus ((-4)EDN) has been solved and refined at atomic resolution (1 A). Two of the extra residues can be placed unambiguously, while the density corresponding to two others is poor. The modified N terminus appears to influence the position of the catalytically important His129, possibly explaining the diminished catalytic activity of this variant. However, (-4)EDN has been shown to be cytotoxic to a Kaposi's sarcoma tumor cell line and other endothelial cell lines. Analysis of the structure and function suggests that the reason for cytotoxicity is most likely due to cellular recognition by the N-terminal extension, since the intrinsic activity of the enzyme is not sufficient for cytotoxicity and the N-terminal extension does not affect the conformation of EDN.     

Centrosymmetric bilayers in the 0.75 A resolution structure of a designed alpha-helical peptide, D,L-Alpha-1.

We report the 0.75 A crystal structure of a racemic mixture of the 12-residue designed peptide "Alpha-1" (Acetyl-ELLKKLLEELKG), the L-enantiomer of which is described in the accompanying paper. Equivalent solutions of the centrosymmetric bilayers were determined by two direct phasing programs in space groups P1 and P1bar. The unit cell contains two L-alpha-helices and two D-alpha-helices. The columnar-sheet bilayer motif seen in L-Alpha-1 is maintained in the D,L-Alpha-1 structure except that each sheet of head-to-tail helices is composed of one enantiomer and is related to its neighboring sheets by inversion symmetry. Comparison to the L-Alpha-1 structure provides further insight into peptide design. The high resolution and small asymmetric unit allowed building an intricate model (R = 13.1%, Rfree = 14.5%) that incorporates much of the discrete disorder of peptide and solvent. Ethanolamine and 2-methyl-2,4-pentanediol (MPD) molecules bind near helix termini. Rigid body analysis identifies sites of restricted displacements and torsions. Side-chain discrete disorder propagates into the backbone of one helix but not the other. Although no side chain in Alpha-1 is rigid, the environments in the crystal restrict some of them to no or only one active torsion.     

The structure and thermal motion of the B800-850 LH2 complex from Rps.acidophila at 2.0A resolution and 100K: new structural features and functionally relevant motions

The structure at 100K of integral membrane light-harvesting complex II (LH2) from Rhodopseudomonas acidophila strain 10050 has been refined to 2.0A resolution. The electron density has been significantly improved, compared to the 2.5A resolution map, by high resolution data, cryo-cooling and translation, libration, screw (TLS) refinement. The electron density reveals a second carotenoid molecule, the last five C-terminal residues of the alpha-chain and a carboxy modified alpha-Met1 which forms the ligand of the B800 bacteriochlorophyll. TLS refinement has enabled the characterisation of displacements between molecules in the complex. B850 bacteriochlorophyll molecules are arranged in a ring of 18 pigments composed of nine approximate dimers. These pigments are strongly coupled and at their equilibrium positions the excited state dipole interaction energies, within and between dimers, are approximately 370cm(-1) and 280cm(-1), respectively. This difference in coupling energy is similar in magnitude to changes in interaction energies arising from the pigment displacements described by TLS tensors. The displacements appear to be non-random in nature and appear to be designed to optimise the modulation of pigment energy interactions. This is the first time that LH2 pigment displacements have been quantified experimentally. The calculated energy changes indicate that there may be significant contributions to inter-pigment energy interactions from molecular displacements and these may be of importance to photosynthetic energy transfer.     

Domain motions of glucosamine-6P synthase: comparison of the anisotropic displacements in the crystals and the catalytic hinge-bending rotation

Glucosamine-6-phosphate synthase channels ammonia over 18 A from glutamine at the glutaminase site to fructose-6P at the synthase site. We have modeled the anisotropic displacements of the glutaminase and synthase domains from the two crystallized states, the enzyme in complex with fructose-6P or in complex with glucose-6P and a glutamine affinity analog, using TLS (rigid-body motion in terms of translation, libration, and screw motions) refinement implemented in REFMAC. The domains displacements in the crystal lattices are compared to the movement of the glutaminase domain relative to the synthase domain that occurs during the catalytic cycle upon glutamine binding, which was visualized by comparing the two structures. This movement was analyzed by the program DYNDOM as a 22.8 degrees rotation around an effective hinge axis running approximately parallel to helix 300-317 of the synthase domain, the glutaminase loop that covers the glutaminase site upon glutamine binding acting as the mechanical hinge.     

Rigid‐body motions of interacting proteins dominate multispecific binding of ubiquitin in a shape‐dependent manner

To understand the dynamic aspects of multispecificity of ubiquitin, we studied nine ubiquitin–ligand (partner protein) complexes by normal mode analysis based on an elastic network model. The coupling between ubiquitin and ligand motions was analyzed by decomposing it into rigid‐body (external) and vibrational (internal) motions of each subunit. We observed that in total the external motions in one of the subunits largely dominated the coupling. The combination of external motions of ubiquitin and the ligands showed general trends of rotations and translations. Moreover, we observed that the rotational motions of ubiquitin were correlated to the ligand orientations. We also identified ubiquitin atomic vibrations that differentiated the orientation of the ligand molecule. We observed that the extents of coupling were correlated to the shapes of the ligands, and this trend was more pronounced when the coupling involved vibrational motions of the ligand. In conclusion, an intricate interplay between internal and external motions of ubiquitin and the ligands help understand the dynamics of multispecificity, which is mostly guided by the shapes of the ligands and the complex. Proteins 2014; 82:77–89. © 2013 Wiley Periodicals, Inc.     

Atomic resolution structures of oxidized [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus in two crystal forms: systematic distortion of [4Fe-4S] cluster in the protein

Diffraction data of two crystal forms (forms I and II) of [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus have been collected to 0.92 A and 1.00 A resolutions, respectively, at 100 K using synchrotron radiation. Anisotropic temperature factors were introduced for all non-hydrogen atoms in the refinement with SHELX-97, in which stereochemical restraints were applied to the protein chain but not to the [4Fe-4S] cluster. The final crystallographic R-factors are 9.8 % for 7.0-0.92 A resolution data of the form I and 11.2 % for the 13.3-1.0 A resolution data of the form II. Many hydrogen atoms as well as multiple conformations for several side-chains have been identified. The present refinement has revised the conformations of several peptide bonds and side-chains assigned previously at 2.3 A resolution; the largest correction was that the main-chain of Pro1 and the side-chain of Lys2 were changed by rotating the C(alpha)-C bond of Lys2. Although the overall structures in the two crystal forms are very similar, conformational differences are observed in the two residues at the middle (Glu29 and Asp30) and the C-terminal residues, which have large temperature factors. The [4Fe-4S] cluster is a distorted cube with non-planar rhombic faces. Slight but significant compression of the four Fe-S bonds along one direction is observed in both crystal forms, and results in the D(2d) symmetry of the cluster. The compressed direction of the cluster relative to the protein is conserved in the two crystal forms and consistent with that in one of the clusters in Clostridium acidurici ferredoxin.     

Dynamic features of homodimer interfaces calculated by normal‐mode analysis

Knowledge of the dynamic features of protein interfaces is necessary for a deeper understanding of protein–protein interactions. We performed normal‐mode analysis (NMA) of 517 nonredundant homodimers and their protomers to characterize dimer interfaces from a dynamic perspective. The motion vector calculated by NMA for each atom of a dimer was decomposed into internal and external motion vectors in individual component subunits, followed by the averaging of time‐averaged correlations between these vectors over atom pairs in the interface. This averaged correlation coefficient (ACC) was defined for various combinations of vectors and investigated in detail. ACCs decrease exponentially with an increasing interface area and r‐value, that is, interface area divided by the entire subunit surface area. As the r‐value reflects the nature of dimer formation, the result suggests that both the interface area and the nature of dimer formation are responsible for the dynamic properties of dimer interfaces. For interfaces with small or medium r‐values and without intersubunit entanglements, ACCs are found to increase on dimer formation when compared with those in the protomer state. In contrast, ACCs do not increase on dimer formation for interfaces with large r‐values and intersubunit entanglements such as in interwinding dimers. Furthermore, relationships between ACCs for intrasubunit atom pairs and for intersubunit atom pairs are found to significantly differ between interwinding and noninterwinding dimers for external motions. External motions are considered as an important factor for characterizing dimer interfaces.     

The X-ray structure of N-methyltryptophan oxidase reveals the structural determinants of substrate specificity

The X-ray structure of monomeric N-methyltryptophan oxidase from Escherichia coli (MTOX) has been solved at 3.2 A resolution by molecular replacement methods using Bacillus sp. sarcosine oxidase structure (MSOX, 43% sequence identity) as search model. The analysis of the substrate binding site highlights the structural determinants that favour the accommodation of the bulky N-methyltryptophan residue in MTOX. In fact, although the nature and geometry of the catalytic residues within the first contact shell of the FAD moiety appear to be virtually superposable in MTOX and MSOX, the presence of a Thr residue in position 239 in MTOX (Met245 in MSOX) located at the entrance of the active site appears to play a key role for the recognition of the amino acid substrate side chain. Accordingly, a 15 fold increase in k(cat) and 100 fold decrease in K(m) for sarcosine as substrate has been achieved in MTOX upon T239M mutation, with a concomitant three-fold decrease in activity towards N-methyltryptophan. These data provide clear evidence for the presence of a catalytic core, common to the members of the methylaminoacid oxidase subfamily, and of a side chain recognition pocket, located at the entrance of the active site, that can be adjusted to host diverse aminoacids in the different enzyme species. The site involved in the covalent attachment of flavin has also been addressed by screening degenerate mutants in the relevant positions around Cys308-FAD linkage. Lys341 appears to be the key residue involved in flavin incorporation and covalent linkage.     

Guest-dependent conformation of 18-crown-6 tetracarboxylic acid: relation to chiral separation of racemic amino acids

(+)-18-crown-6 tetracarboxylic acid (18C6H(4)) has been used as a chiral selector for various amines and amino acids. To further clarify the structural scaffold of 18C6H(4) for chiral separation, single crystal X-ray analysis of its glycine(+) (1), H3O+ (2), H5O2+ (3), NH4+ (4), and 2CH3NH3+ (5) complexes was performed and the guest-dependent conformation of 18C6H(4) was investigated. The crown ether ring of 18C6H4 in 3, 4, and 5 took a symmetrical C2 or C2-like conformation, whereas that in 1 and 2 took an asymmetric C1 conformation, which is commonly observed in complexes with various optically active amino acids. The overall survey of the present and related complexes suggests that the molecular conformation of 18C6H4 is freely changeable within an allowable range, depending on the molecular shape and interaction mode with the cationic guest. On the basis of the present results, we propose the allowable conformational variation of 18C6H4 and a possible transition pathway from its primary conformation to the conformation suitable for chiral separation of racemic amines and amino acids.     

Crystal structure of the N domain of Lon protease from Mycobacterium avium complex

Lon protease is evolutionarily conserved in prokaryotes and eukaryotic organelles. The primary function of Lon is to selectively degrade abnormal and certain regulatory proteins to maintain the homeostasis in vivo. Lon mainly consists of three functional domains and the N‐terminal domain is required for the substrate selection and recognition. However, the precise contribution of the N‐terminal domain remains elusive. Here, we determined the crystal structure of the N‐terminal 192‐residue construct of Lon protease from Mycobacterium avium complex at 2.4 å resolution,and measured NMR‐relaxation parameters of backbones. This structure consists of two subdomains, the β‐strand rich N‐terminal subdomain and the five‐helix bundle of C‐terminal subdomain, connected by a flexible linker,and is similar to the overall structure of the N domain of Escherichia coli Lon even though their sequence identity is only 26%. The obtained NMR‐relaxation parameters reveal two stabilized loops involved in the structural packing of the compact N domain and a turn structure formation. The performed homology comparison suggests that structural and sequence variations in the N domain may be closely related to the substrate selectivity of Lon variants. Our results provide the structure and dynamics characterization of a new Lon N domain, and will help to define the precise contribution of the Lon N‐terminal domain to the substrate recognition.     

Packing analysis and the crystal chemistry of 2,5-dibenzylidenecyclopent-3-ene-1-one. Rationale for a non-topochemical solid-state reaction

The molecular and crystal structure of 2,5-dibenzylidenecyclopent-3-ene-1-one has been studied in detail to explain the formation of a non-topochemical pseudo-mirror-symmetric dimer upon photoirradiation. Packing energy calculations, analysis of the thermal motion, and lattice energy calculations are employed to analyse and understand the observed dimerization reaction, crystal structure, and crystal properties. Supplementary data relating to this article have been deposited with the British Library as Supplementary Publication No. SUP 82137 (6 pages).     

Crystal structure of the EF-hand parvalbumin at atomic resolution (0.91 A) and at low temperature (100 K). Evidence for conformational multistates within the hydrophobic core.

Several crystal structures of parvalbumin (Parv), a typical EF-hand protein, have been reported so far for different species with the best resolution achieving 1.5 A. Using a crystal grown under microgravity conditions, cryotechniques (100 K), and synchrotron radiation, it has now been possible to determine the crystal structure of the fully Ca2+-loaded form of pike (component pI 4.10) Parv.Ca2 at atomic resolution (0.91 A). The availability of such a high quality structure offers the opportunity to contribute to the definition of the validation tools useful for the refinement of protein crystal structures determined to lower resolution. Besides a better definition of most of the elements in the protein three-dimensional structure than in previous studies, the high accuracy thus achieved allows the detection of well-defined alternate conformations, which are observed for 16 residues out of 107 in total. Among them, six occupy an internal position within the hydrophobic core and converge toward two small buried cavities with a total volume of about 60 A3. There is no indication of any water molecule present in these cavities. It is probable that at temperatures of physiological conditions there is a dynamic interconversion between these alternate conformations in an energy-barrier dependent manner. Such motions for which the amplitudes are provided by the present study will be associated with a time-dependent remodeling of the void internal space as part of a slow dynamics regime (millisecond timescales) of the parvalbumin molecule. The relevance of such internal dynamics to function is discussed.     

The three‐dimensional structure of TrmB,a transcriptional regulator of dual function in the hyperthermophilic archaeon Pyrococcus furiosus in complex with sucrose

TrmB is a repressor that binds maltose, maltotriose, and sucrose, as well as other α‐glucosides. It recognizes two different operator sequences controlling the TM (Trehalose/Maltose) and the MD (Maltodextrin) operon encoding the respective ABC transporters and sugar‐degrading enzymes. Binding of maltose to TrmB abrogates repression of the TM operon but maintains the repression of the MD operon. On the other hand, binding of sucrose abrogates repression of the MD operon but maintains repression of the TM operon. The three‐dimensional structure of TrmB in complex with sucrose was solved and refined to a resolution of 3.0 Å. The structure shows the N‐terminal DNA binding domain containing a winged‐helix‐turn‐helix (wHTH) domain followed by an amphipathic helix with a coiled‐coil motif. The latter promotes dimerization and places the symmetry mates of the putative recognition helix in the wHTH motif about 30 Å apart suggesting a canonical binding to two successive major grooves of duplex palindromic DNA. This suggests that the structure resembles the conformation of TrmB recognizing the pseudopalindromic TM promoter but not the conformation recognizing the nonpalindromic MD promoter.     

Crystal structure of human carbonic anhydrase XIII and its complex with the inhibitor acetazolamide

The cytosolic isoform XIII is a recently discovered member of the human carbonic anhydrase (hCA, EC 4.2.1.1) family. It is selectively expressed among other tissues in the reproductive organs, where it may control pH and ion balance regulation, ensuring thus proper fertilization conditions. The authors report here the X-ray crystallographic structure of this isozyme in the unbound state and in complex with a classical sulfonamide inhibitor, namely acetazolamide. A detailed comparison of the obtained structural data with those already reported for other CA isozymes provides novel insights into the catalytic properties of the members of this protein family. On the basis of the inhibitory properties of acetazolamide against various cytosolic/transmembrane isoforms and the structural differences detected within the active site of the various CA isoforms, further prospects for the design of isozyme-specific CA inhibitors are here proposed.