Changes in interactions in complexes of hirudin derivatives and human alpha-thrombin due to different crystal forms.

The three-dimensional structures of D-Phe-Pro-Arg-chloromethyl ketone-inhibited thrombin in complex with Tyr-63-sulfated hirudin (ternary complex) and of thrombin in complex with the bifunctional inhibitor D-Phe-Pro-Arg-Pro-(Gly)4-hirudin (CGP 50,856, binary complex) have been determined by X-ray crystallography in crystal forms different from those described by Skrzypczak-Jankun et al. (Skrzypczak-Jankun, E., Carperos, V.E., Ravichandran, K.G., & Tulinsky, A., 1991, J. Mol. Biol. 221, 1379-1393). In both complexes, the interactions of the C-terminal hirudin segments of the inhibitors binding to the fibrinogen-binding exosite of thrombin are clearly established, including residues 60-64, which are disordered in the earlier crystal form. The interactions of the sulfate group of Tyr-63 in the ternary complex structure explain why natural sulfated hirudin binds with a 10-fold lower K(i) than the desulfated recombinant material. In this new crystal form, the autolysis loop of thrombin (residues 146-150), which is disordered in the earlier crystal form, is ordered due to crystal contacts. Interactions between the C-terminal fragment of hirudin and thrombin are not influenced by crystal contacts in this new crystal form, in contrast to the earlier form. In the bifunctional inhibitor-thrombin complex, the peptide bond between Arg-Pro (P1-P1') seems to be cleaved.     

Crystal structure of the unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate.

The crystal structure of unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, was determined to 2.7 A resolution by X-ray crystallography. The transition state analog binds at the active site in an extended conformation. As compared to the binding of the same analog in the activated enzyme, the analog binds in a reverse orientation. The active site Lys 201 is within hydrogen bonding distance of the carboxyl oxygen of the analog. Loop 6 (residues 330-339) remains open and flexible upon binding of the analog in the unactivated enzyme, in contrast to the closed and ordered loop 6 in the activated enzyme complex. The transition state analog is exposed to solvent due to the open conformation of loop 6.     

Crystal structure of cyclophilin A complexed with a binding site peptide from the HIV-1 capsid protein.

The cellular protein, cyclophilin A (CypA), is incorporated into the virion of the type 1 human immunodeficiency virus (HIV-1) via a direct interaction with the capsid domain of the viral Gag polyprotein. We demonstrate that the capsid sequence 87His-Ala-Gly-Pro-Ile-Ala92 (87HAGPIA92) encompasses the primary cyclophilin A binding site and present an X-ray crystal structure of the CypA/HAGPIA complex. In contrast to the cis prolines observed in all previously reported structures of CypA complexed with model peptides, the proline in this peptide, Pro 90, binds the cyclophilin A active site in a trans conformation. We also report the crystal structure of a complex between CypA and the hexapeptide HVGPIA, which also maintains the trans conformation. Comparison with the recently determined structures of CypA in complexes with larger fragments of the HIV-1 capsid protein demonstrates that CypA recognition of these hexapeptides involves contacts with peptide residues Ala(Val) 88, Gly 89, and Pro 90, and is independent of the context of longer sequences.     

Charting the surfaces of the purple membrane

The preponderance of structural data of the purple membrane from X-ray diffraction (XRD), electron crystallography (EC), and atomic force microscopy (AFM) allows us to ask questions about the structure of bacteriorhodopsin itself, as well as about the information derived from the different techniques. The transmembrane helices of bacteriorhodopsin are quite similar in both EC and XRD models. In contrast, the loops at the surfaces of the purple membrane show the highest variability between the atomic models, comparable to the height variance measured by AFM. The excellent agreement of the AFM topographs with the atomic models from XRD builds confidence in the results. Small technical difficulties in EC lead to poorer resolution of the loop structures, although the combination of atomic models with AFM surfaces allows clear interpretation of the extent and flexibility of the loop structures. While XRD remains the premier technique to determine very-high-resolution structures, EC offers a method to determine loop structures unhindered by three-dimensional crystal contacts, and AFM provides information about surface structures and their flexibility under physiological conditions.     

The Overall Architecture and Receptor Binding of Pneumococcal Carbohydrate-Antigen-Hydrolyzing Enzymes

The TIGR4 and SP3-BS71 strains of Streptococcus pneumoniae each produce family 98 glycoside hydrolases, called Sp4GH98 and Sp3GH98, respectively, which have different modular architectures and substrate specificities. Sp4GH98 degrades the Lewis^Y antigen and possesses three C-terminal family 47 carbohydrate-binding modules (CBMs) that bind to this substrate. Sp3GH98 degrades the blood group A/B antigens and has two N-terminal family 51 CBMs that are of unknown function. Here, we examine the complex carbohydrate-binding specificity of the family 51 CBMs from Sp3GH98 (referred to as CBM51-1 and CBM51-2), the structural basis of this interaction, and the overall solution conformations of both Sp3GH98 and Sp4GH98, which are shown to be fully secreted proteins. Through glycan microarray binding analysis and isothermal titration calorimetry, CBM51-1 is found to bind specifically to the blood group A/B antigens. However, due to a series of relatively small structural rearrangements that were revealed in structures determined by X-ray crystallography, CBM51-2 appears to be incapable of binding carbohydrates. Analysis of small-angle X-ray scattering data in combination with the available high-resolution X-ray crystal structures of the Sp3GH98 and Sp4GH98 catalytic modules and their CBMs yielded models of the biological solution structures of the full-length enzymes. These studies reveal the complex architectures of the two enzymes and suggest that carbohydrate recognition by the CBMs and the activity of the catalytic modules are not directly coupled.     

Conformational sampling of the botulinum neurotoxin serotype A light chain: implications for inhibitor binding

Botulinum neurotoxins (BoNTs) are the most potent of the known biological toxins, and consequently are listed as category A biowarfare agents. Currently, the only treatments against BoNTs include preventative antitoxins and long-term supportive care. Consequently, there is an urgent need for therapeutics to counter these enzymes--post exposure. In a previous study, we identified a number of small, nonpeptidic lead inhibitors of BoNT serotype A light chain (BoNT/A LC) metalloprotease activity, and we identified a common pharmacophore for these molecules. In this study, we have focused on how the dynamic movement of amino acid residues in and surrounding the substrate binding cleft of the BoNT/A LC might affect inhibitor binding modes. The X-ray crystal structures of two BoNT/A LCs (PDB refcodes=3BTA and 1E1H) were examined. Results from these analyses indicate that the core structural features of the examined BoNT/A LCs, including alpha-helices and beta-sheets, remained relatively unchanged during 1 ns dynamics trajectories. However, conformational flexibility was observed in surface loops bordering the substrate binding clefts in both examined structures. Our analyses indicate that these loops may possess the ability to decrease the solvent accessibility of the substrate binding cleft, while at the same time creating new residue contacts for the inhibitors. Loop movements and conformational/positional analyses of residues within the substrate binding cleft are discussed with respect to BoNT/A LC inhibitor binding and our common pharmacophore for inhibition. The results from these studies may aid in the future identification/development of more potent small molecule inhibitors that take advantage of new binding contacts in the BoNT/A LC.     

Trapping intermediates in the crystal: ligand binding to myoglobin

Crystal structures of the reactive short-lived species that occur in chemical or binding reactions can be determined using X-ray crystallography via time-resolved or kinetic trapping approaches. Recently, various kinetic trapping methods have been used to determine the structure of intermediates in ligand binding to myoglobin.     

Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine

The Methanococcus jannaschii tRNA(Tyr)/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-L-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 A, respectively, for comparison with the published structure of TyrRS complexed with tRNA(Tyr) and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257-263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through pi-stacking and hydrogen bonding interactions. Loop 133-143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNA(Tyr). Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133-143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over L-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids.     

Molecular basis for dimer formation of TRbeta variant D355R

Protein quality and stability are critical during protein purification for X-ray crystallography. A target protein that is easy to manipulate and crystallize becomes a valuable product useful for high-throughput crystallography for drug design and discovery. In this work, a single surface mutation, D355R, was shown to be crucial for converting the modestly stable monomeric ligand binding domain of the human thyroid hormone receptor (TR LBD) into a stable dimer. The structure of D335R TR LBD mutant was solved using X-ray crystallography and refined to 2.2 A resolution with R(free)/R values of 24.5/21.7. The crystal asymmetric unit reveals the TR dimer with two molecules of the hormone-bound LBD related by twofold symmetry. The ionic interface between the two LBDs comprises residues within loop H10-H11 and loop H6-H7 as well as the C-terminal halves of helices 8 of both protomers. Direct intermolecular contacts formed between the introduced residue Arg 355 of one TR molecule and Glu 324 of the second molecule become a part of the extended dimerization interface of 1330 A(2) characteristic for a strong complex assembly that is additionally strengthened by buffer solutes.     

Crystallographic determination of reduced bovine superoxide dismutase at pH 5.0 and of anion binding to its active site

The crystal structures of dithionite-reduced bovine Cu(I),Zn superoxide dismutase and of its adducts with the inorganic anions azide and thyocyanide have been determined in a C222₁ crystal form obtained at pH?5.0. This crystal form is characterized by a high solvent content (72%) and by having the two Cu,ZnSOD monomers (A and B) in different crystal environments. One of them (B) is involved in few intermolecular crystal contacts so that it is in a more "solution like" environment, as indicated by average temperature factors which are about twice those of the other monomer. The differences in crystal packing affect the active site structures. While in the A monomer the Cu(I) is coordinated to all four histidine residues, in the B monomer the bridging His61 side chain is found disordered, implying partial detachment from copper. The same effect occurs in the structures of the anion complexes. The inorganic anions are found bound in the active site cavity, weakly interacting with copper at distances ranging from 2.5 to 2.8?Å. The copper site in the A subunit of the native enzyme structure displays significant electron density resembling a diatomic molecule, bound side-on at about 2.8?Å from the metal, which cannot be unambiguously interpreted. The crystallographic data suggest that the existence of the His61 bridge between copper and zinc is dominated by steric more than electronic factors and that the solution state favors the His61 detachment. These structures confirm the existence of an energetically available state for Cu(I) in Cu,ZnSOD where the histidinato bridge to zinc is maintained. This state appears to be favored by tighter crystal contacts. The binding of the anions in the active site cavity is different from that observed in the oxidized enzyme and it appears to be dominated by electrostatic interactions within the cavity. The anion binding mode observed may model the substrate interaction with the reduced enzyme during catalysis.     

Conformations of kinesin: solution vs. crystal structures and interactions with microtubules

Recently, the molecular structures of monomeric and dimeric kinesin constructs in complex with ADP have been determined by X-ray crystallography (Kull et al. 1996; Kozielski et al. 1997 a; Sack et al. 1997). The “motor” or “head” domains have almost identical conformations in the known crystal structures, yet the kinesin dimer is asymmetric: the orientation of the two heads relative to the coiled-coil formed by their neck regions is different. We used small angle solution scattering of kinesin constructs and microtubules decorated with kinesin in order to find out whether these crystal structures are of relevance for kinesin's structure under natural conditions and for its interaction with microtubules. Our preliminary results indicate that the crystal structures of monomeric and dimeric kinesin are similar to their structures in solution, though in solution the center-of-mass distance between the motor domains of the dimer could be slightly greater. The crystal structure of dimeric kinesin can be interpreted as representing two equivalent conformations. Transitions between these or very similar conformational states may occur in solution. Binding of kinesin to microtubules has conformational effects on both, the kinesin and the microtubule. Solution scattering of kinesin decorated microtubules reveals a peak in intensity that is characteristic for the B-surface lattice and that can be used to monitor the axial repeat of the microtubules under various conditions. In decoration experiments, dimeric kinesin dissociates, at least partly, leading to a stoichiometry of 1:1 (one kinesin head per tubulin dimer; Thormählen et al. 1998 a) in contrast to the stoichiometry of 2:1 reported for dimeric ncd. This discrepancy is possibly due to the effect of steric hindrance between kinesin dimers on adjacent binding sites.     

Crystal contact-free conformation of an intrinsically flexible loop in protein crystal: Tim21 as the case study

BackgroundIn protein crystals, flexible loops are frequently deformed by crystal contacts, whereas in solution, the large motions result in the poor convergence of such flexible loops in NMR structure determinations. We need an experimental technique to characterize the structural and dynamic properties of intrinsically flexible loops of protein molecules.MethodsWe designed an intended crystal contact-free space (CCFS) in protein crystals, and arranged the flexible loop of interest in the CCFS. The yeast Tim 21 protein was chosen as the model protein, because one of the loops (loop 2) is distorted by crystal contacts in the conventional crystal.ResultsYeast Tim21 was fused to the MBP protein by a rigid α-helical linker. The space created between the two proteins was used as the CCFS. The linker length provides adjustable freedom to arrange loop 2 in the CCFS. We re-determined the NMR structure of yeast Tim21, and conducted MD simulations for comparison. Multidimensional scaling was used to visualize the conformational similarity of loop 2. We found that the crystal contact-free conformation of loop 2 is located close to the center of the ensembles of the loop 2 conformations in the NMR and MD structures.ConclusionsLoop 2 of yeast Tim21 in the CCFS adopts a representative, dominant conformation in solution.General significanceNo single powerful technique is available for the characterization of flexible structures in protein molecules. NMR analyses and MD simulations provide useful, but incomplete information. CCFS crystallography offers a third route to this goal.     

History of protein crystallography in China

China has a strong background in X-ray crystallography dating back to the 1920s. Protein crystallography research in China was first developed following the successful synthesis of insulin in China in 1966. The subsequent determination of the three-dimensional structure of porcine insulin made China one of the few countries which could determine macromolecular structures by X-ray diffraction methods in the late 1960s and early 1970s. After a slow period during the 1970s and 1980s, protein crystallography in China has reached a new climax with a number of outstanding accomplishments. Here, I review the history and progress of protein crystallography in China and detail some of the recent research highlights, including the crystal structures of two membrane proteins as well as the structural genomics initiative in China.     

The contribution of metal ions to the conformational stability of ribonuclease T1: crystal versus solution.

In the crystalline state, ribonuclease T1 binds calcium ions at different lattice-dependent positions. In solution, its conformational stability is also remarkably increased in the presence of divalent metal ions. Combining urea unfolding studies and X-ray crystallography, we compared the presence of several metal ions at specific sites in the protein to their contribution to the overall stabilizing effect in solution. We constructed thermodynamic cycles involving particular metal ions and specific carboxylate functions. The resulting coupling energies indicate that some (but not all) metal ions found at lattice contacts in crystal structures may indeed significantly contribute to stability enhancement in the presence of metal ions in solution.     

Structural Analysis of HMGD-DNA Complexes Reveals Influence of Intercalation on Sequence Selectivity and DNA Bending

The ubiquitous, eukaryotic, high-mobility group box (HMGB) chromosomal proteins promote many chromatin-mediated cellular activities through their non-sequence-specific binding and bending of DNA. Minor-groove DNA binding by the HMG box results in substantial DNA bending toward the major groove owing to electrostatic interactions, shape complementarity, and DNA intercalation that occurs at two sites. Here, the structures of the complexes formed with DNA by a partially DNA intercalation-deficient mutant of Drosophila melanogaster HMGD have been determined by X-ray crystallography at a resolution of 2.85 Å. The six proteins and 50 bp of DNA in the crystal structure revealed a variety of bound conformations. All of the proteins bound in the minor groove, bridging DNA molecules, presumably because these DNA regions are easily deformed. The loss of the primary site of DNA intercalation decreased overall DNA bending and shape complementarity. However, DNA bending at the secondary site of intercalation was retained and most protein-DNA contacts were preserved. The mode of binding resembles the HMGB1 box A-cisplatin-DNA complex, which also lacks a primary intercalating residue. This study provides new insights into the binding mechanisms used by HMG boxes to recognize varied DNA structures and sequences as well as modulate DNA structure and DNA bending.     

Microcrystallization techniques for serial femtosecond crystallography using photosystem II from Thermosynechococcus elongatus as a model system

Serial femtosecond crystallography (SFX) is a new emerging method, where X-ray diffraction data are collected from a fully hydrated stream of nano- or microcrystals of biomolecules in their mother liquor using high-energy, X-ray free-electron lasers. The success of SFX experiments strongly depends on the ability to grow large amounts of well-ordered nano/microcrystals of homogeneous size distribution. While methods to grow large single crystals have been extensively explored in the past, method developments to grow nano/microcrystals in sufficient amounts for SFX experiments are still in their infancy. Here, we describe and compare three methods (batch, free interface diffusion (FID) and FID centrifugation) for growth of nano/microcrystals for time-resolved SFX experiments using the large membrane protein complex photosystem II as a model system.     

What is the average conformation of bacteriophage T4 lysozyme in solution? A domain orientation study using dipolar couplings measured by solution NMR

Lysozyme from T4 bacteriophage is comprised of two domains that are both involved in binding substrate. Although wild-type lysozyme has been exclusively crystallized in a closed form that is similar to the peptidoglycan-bound conformation, a more open structure is thought to be required for ligand binding. To determine the relative arrangement of domains within T4 lysozyme in the solution state, dipolar couplings were measured in several different dilute liquid crystalline media by solution NMR methods. The dipolar coupling data were analyzed with a domain orientation procedure described previously that utilizes high- resolution X-ray structures. The cleft between the domains is significantly larger in the average solution structure than what is observed in the X-ray structure of the ligand-free form of the protein (approximately 17 degrees closure from solution to X-ray structures). A comparison of the solution domain orientation with X-ray-derived structures in the protein data base shows that the solution structure resembles a crystal structure obtained for the M6I mutant. Dipolar couplings were also measured on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-domain disulfide bond (T4SS). In this case, the inter-domain solution structure was found to be more closed than was observed in the crystal (approximately 11 degrees). Direct refinement of lysozyme crystal structures with the measured dipolar couplings using the program CNS, establishes that this degree of closure can be accommodated whilst maintaining the inter-domain cystine bond. The differences between the average solution conformations obtained using dipolar couplings and the crystal conformations for both forms of lysozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and the importance of alternative methods to X-ray crystallography for evaluating inter-domain structure.     

Carbonic Anhydrase Inhibitors: X-ray Crystallographic Structure of the Adduct of Human Isozyme II with the Perfluorobenzoyl Analogue of Methazolamide. Implications for the Drug Design of Fluorinated Inhibitors

The X-ray crystal structure for the adduct of human carbonic anhydrase (hCA) II with 4-methyl-5-perfluorophenylcarboximido-δ2-1,3,4-thiadiazoline-2-sulfonamide (PFMZ), a topically acting antiglaucoma sulfonamide, has been resolved at a resolution of 1.8?Å. This compound is almost 10 times more effective as a hCA II inhibitor (KI of 1.5?nM) compared to the lead molecule, methazolamide, a clinically used drug (KI of 14?nM). Its binding to the enzyme active site is similar to that of other sulfonamide inhibitors, considering the interactions of the sulfonamide zinc anchoring group and thiadiazoline ring contacts, but differs considerably when the perfluorobenzoylimino fragment of the molecule is analyzed. Indeed, several unprecedented strong hydrogen bonds involving the imino nitrogen, carbonyl oxygen, a fluorine atom in the ortho position of the inhibitor, and two water molecules, as well as Gln 92 of the enzyme active site were seen. A stacking interaction of the perfluorophenyl ring of the inhibitor and the aromatic ring of Phe 131 was also observed for the first time in a CA–sulfonamide adduct. All these findings prove that more potent CA inhibitors incorporating perfluoroaryl/alkyl tails may be designed, with potentially improved antiglaucoma properties, in view of the new types of interactions seen here between the enzyme and the perfluorobenzoylated analogue of methazolamide.     

Alternate modes of binding in two crystal structures of alkaline phosphatase-inhibitor complexes

Two high resolution crystal structures of Escherichia coli alkaline phosphatase (AP) in the presence of phosphonate inhibitors are reported. The phosphonate compounds, phosphonoacetic acid (PAA) and mercaptomethylphosphonic acid (MMP), bind competitively to AP with dissociation constants of 5.5 and 0.6 mM, respectively. The structures of the complexes of AP with PAA and MMP were refined at high resolution to crystallographic R-values of 19.0 and 17.5%, respectively. Refinement of the AP-inhibitor complexes was carried out using X-PLOR. The final round of refinement was done using SHELXL-97. Crystallographic analyses of the inhibitor complexes reveal different binding modes for the two phosphonate compounds. The significant difference in binding constants can be attributed to these alternative binding modes observed in the high resolution X-ray structures. The phosphinyl group of PAA coordinates to the active site zinc ions in a manner similar to the competitive inhibitor and product inorganic phosphate. In contrast, MMP binds with its phosphonate moiety directed toward solvent. Both enzyme-inhibitor complexes exhibit close contacts, one of which has the chemical and geometrical potential to be considered an unconventional hydrogen bond of the type C-H...X.     

Calcium ion exchange in crystalline gelsolin

Gelsolin is a calcium and pH-sensitive modulator of actin filament length. Here, we use X-ray crystallography to examine the extraction and exchange of calcium ions from their binding sites in different crystalline forms of the activated N and C-terminal halves of gelsolin, G1-G3 and G4-G6, respectively. We demonstrate that the combination of calcium and low pH activating conditions do not induce conformational changes in G4-G6 beyond those elicited by calcium alone. EGTA is able to remove calcium ions bound to the type I and type II metal ion-binding sites in G4-G6. Constrained by crystal contacts and stabilized by interdomain interaction surfaces, the gross structure of calcium-depleted G4-G6 remains that of the activated form. However, high-resolution details of changes in the ion-binding sites may represent the initial steps toward restoration of the arrangement of domains found in the calcium-free inactive form of gelsolin in solution. Furthermore, bathing crystals with the trivalent calcium ion mimic, Tb3+, results in anomalous scattering data that permit unequivocal localization of terbium ions in each of the proposed type I and type II ion-binding sites of both halves of gelsolin. In contrast to predictions based on solution studies, we find that no calcium ion is immune to exchange.