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1.
The molecular basis of fructose‐2,6‐bisphosphatase (F‐2,6‐P2ase) of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB) was investigated using the crystal structures of the human inducible form (PFKFB3) in a phospho‐enzyme intermediate state (PFKFB3‐P?F‐6‐P), in a transition state–analogous complex (PFKFB3?AlF4), and in a complex with pyrophosphate (PFKFB3?PPi) at resolutions of 2.45, 2.2, and 2.3 Å, respectively. Trapping the PFKFB3‐P?F‐6‐P intermediate was achieved by flash cooling the crystal during the reaction, and the PFKFB3?AlF4 and PFKFB3?PPi complexes were obtained by soaking. The PFKFB3?AlF4 and PFKFB3?PPi complexes resulted in removing F‐6‐P from the catalytic pocket. With these structures, the structures of the Michaelis complex and the transition state were extrapolated. For both the PFKFB3‐P formation and break down, the phosphoryl donor and the acceptor are located within ～5.1 Å, and the pivotal point 2‐P is on the same line, suggesting an “in‐line” transfer with a direct inversion of phosphate configuration. The geometry suggests that NE2 of His253 undergoes a nucleophilic attack to form a covalent N? P bond, breaking the 2O? P bond in the substrate. The resulting high reactivity of the leaving group, 2O of F‐6‐P, is neutralized by a proton donated by Glu322. Negative charges on the equatorial oxygen of the transient bipyramidal phosphorane formed during the transfer are stabilized by Arg252, His387, and Asn259. The C‐terminal domain (residues 440–446) was rearranged in PFKFB3?PPi, implying that this domain plays a critical role in binding of substrate to and release of product from the F‐2,6‐P2ase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction. Proteins 2012; © 2011 Wiley Periodicals, Inc. 相似文献
2.
Alvizo O Mittal S Mayo SL Schiffer CA 《Protein science : a publication of the Protein Society》2012,21(7):1029-1041
HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease. 相似文献
3.
Yoshiaki Kiso Hikaru Matsumoto Sota Mizumoto Tooru Kimura Yoichi Fujiwara Kenichi Akaji 《Peptide Science》1999,51(1):59-68
The human immunodeficiency virus (HIV) codes for an aspartic protease known to be essential for retroviral maturation and replication. HIV protease is formed from two identical 99 amino acid peptides. We synthesized [(NHCH2CH2—S—CH2CO)51–52, Ala67,95]HIV‐1 protease using the thioether chemical ligation method, and then prepared the [(NHCH2CH2—S—CH2CO)51–52, Ala67,95, Cys98]HIV‐1 protease dimer analogue covalently linked by a disulfide bridge. These HIV‐1 protease analogues effectively cleaved the Tyr–Phe‐type substrate, but had weak affinity to the Tyr–Pro‐type substrate. Consequently, the molecular recognition of the protease analogues differs from that of the wild‐type enzyme. Based on the substrate transition state, we designed and synthesized a novel class of HIV protease inhibitors containing an unnatural amino acid, (2S, 3S)‐3‐amino‐2‐hydroxy‐4‐phenylbutyric acid, named allophenylnorstatine, with a hydroxymethylcarbonyl (HMC) isostere. The stereochemistry of the hydroxyl group was significant for the enzyme inhibition and the HMC group interacted excellently with the aspartic acid carboxyl groups of HIV protease active site in the essentially same hydrogen‐bonding mode as the transition state. Small dipeptide‐based HIV protease inhibitors containing the HMC isostere were studied as advantageous compounds. Among them, a dipeptide‐based HIV protease inhibitor, KNI‐577, exhibited potent antiviral activities, low cytotoxicity, and good pharmacokinetic properties. © 1999 John Wiley & Sons, Inc. Biopoly 51: 59–68, 1999 相似文献
4.
Kathleen M. Clark Jermaine L. Jenkins Nadia Fedoriw Mark E. Dumont 《Protein science : a publication of the Protein Society》2017,26(2):242-257
The function and localization of proteins and peptides containing C‐terminal “CaaX” (Cys‐aliphatic‐aliphatic‐anything) sequence motifs are modulated by post‐translational attachment of isoprenyl groups to the cysteine sulfhydryl, followed by proteolytic cleavage of the aaX amino acids. The zinc metalloprotease ZMPSTE24 is one of two enzymes known to catalyze this cleavage. The only identified target of mammalian ZMPSTE24 is prelamin A, the precursor to the nuclear scaffold protein lamin A. ZMPSTE24 also cleaves prelamin A at a second site 15 residues upstream from the CaaX site. Mutations in ZMPSTE24 result in premature‐aging diseases and inhibition of ZMPSTE24 activity has been reported to be an off‐target effect of HIV protease inhibitors. We report here the expression (in yeast), purification, and crystallization of human ZMPSTE24 allowing determination of the structure to 2.0 Å resolution. Compared to previous lower resolution structures, the enhanced resolution provides: (1) a detailed view of the active site of ZMPSTE24, including water coordinating the catalytic zinc; (2) enhanced visualization of fenestrations providing access from the exterior to the interior cavity of the protein; (3) a view of the C‐terminus extending away from the main body of the protein; (4) localization of ordered lipid and detergent molecules at internal and external surfaces and also projecting through fenestrations; (5) identification of water molecules associated with the surface of the internal cavity. We also used a fluorogenic assay of the activity of purified ZMPSTE24 to demonstrate that HIV protease inhibitors directly inhibit the human enzyme in a manner indicative of a competitive mechanism. 相似文献
5.
Caseinolytic proteases are large oligomeric assemblies responsible for maintaining protein homeostasis in bacteria and in so doing influence a wide range of biological processes. The functional assembly involves three chaperones together with the oligomeric caseinolytic protease catalytic subunit P (ClpP). This protease represents a potential target for therapeutic intervention in pathogenic bacteria. Here, we detail an efficient protocol for production of recombinant ClpP from Francisella tularensis, and the structural characterization of three crystal forms which grow under similar conditions. One crystal form reveals a compressed state of the ClpP tetradecamer and two forms an open state. A comparison of the two types of structure infers that differences at the enzyme active site result from a conformational change involving a highly localized disorder‐order transition of a β‐strand α‐helix combination. This transition occurs at a subunit‐subunit interface. Our study may now underpin future efforts in a structure‐based approach to target ClpP for inhibitor or activator development. Proteins 2016; 85:188–194. © 2016 Wiley Periodicals, Inc. 相似文献
6.
The structural and functional role of conserved residue G86 in HIV‐1 protease (PR) was investigated by NMR and crystallographic analyses of substitution mutations of glycine to alanine and serine (PRG86A and PRG86S). While PRG86S had undetectable catalytic activity, PRG86A exhibited ～6000‐fold lower catalytic activity than PR. 1H‐15N NMR correlation spectra revealed that PRG86A and PRG86S are dimeric, exhibiting dimer dissociation constants (Kd) of ～0.5 and ～3.2 μM, respectively, which are significantly lower than that seen for PR with R87K mutation (Kd > 1 mM). Thus, the G86 mutants, despite being partially dimeric under the assay conditions, are defective in catalyzing substrate hydrolysis. NMR spectra revealed no changes in the chemical shifts even in the presence of excess substrate, indicating very poor binding of the substrate. Both NMR chemical shift data and crystal structures of PRG86A and PRG86S in the presence of active‐site inhibitors indicated high structural similarity to previously described PR/inhibitor complexes, except for specific perturbations within the active site loop and around the mutation site. The crystal structures in the presence of the inhibitor showed that the region around residue 86 was connected to the active site by a conserved network of hydrogen bonds, and the two regions moved further apart in the mutants. Overall, in contrast to the role of R87 in contributing significantly to the dimer stability of PR, G86 is likely to play an important role in maintaining the correct geometry of the active site loop in the PR dimer for substrate binding and hydrolysis. Proteins 2010. © 2009 Wiley‐Liss, Inc. 相似文献
7.
We report a clustering of public human protein kinase structures based on the conformations of two structural elements, the activation segment and the C-helix, revealing three discrete clusters. One cluster includes kinases in catalytically active conformations. Each of the other clusters contains a distinct inactive conformation. Typically, kinases adopt at most one of the inactive conformations in available X-ray structures, implying that one of the conformations is preferred for many kinases. The classification is consistent with selectivity profiles of several well-characterized kinase inhibitors. We show further that inhibitor selectivity profiles guide kinase classification. For example, selective inhibition of lck among src-family kinases by imatinib (Gleevec) suggests that the relative stabilities of inactive conformations of lck are different from other src-family kinases. We report the X-ray structure of the lck/imatinib complex, confirming that the conformation adopted by lck is distinct from other structurally-characterized src-family kinases and instead resembles kinases abl1 and kit in complex with imatinib. Our classification creates new paths for designing small-molecule inhibitors. 相似文献
8.
《Acta Crystallographica. Section D, Structural Biology》2017,73(10):822-828
The structural and biochemical characterization of broadly neutralizing anti‐HIV‐1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV‐1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N‐glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high‐mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high‐resolution structures. 3.5 and 3.9 Å resolution crystal structures of the HIV‐1 Env trimer with fully processed and native glycosylation were solved, revealing a glycan shield of high‐mannose and complex‐type N‐glycans that were used to define the complete epitopes of two bNAbs. Here, the refinement of the N‐glycans in the crystal structures is discussed and comparisons are made with glycan densities in glycosylated Env structures derived by single‐particle cryo‐electron microscopy. 相似文献
9.
Fiana Burshteyn Michael Cassidy Ebony Gary Brandan Hillerich Zhong‐Ke Yao Paul R. Carlier Maxim Totrov James D. Love 《Proteins》2015,83(12):2124-2136
Coxiella burnetii is a highly infectious bacterium and potential agent of bioterrorism. However, it has not been studied as extensively as other biological agents, and very few of its proteins have been structurally characterized. To address this situation, we undertook a study of critical metabolic enzymes in C. burnetii that have great potential as drug targets. We used high‐throughput techniques to produce novel crystal structures of 48 of these proteins. We selected one protein, C. burnetii dihydrofolate reductase (CbDHFR), for additional work to demonstrate the value of these structures for structure‐based drug design. This enzyme's structure reveals a feature in the substrate binding groove that is different between CbDHFR and human dihydrofolate reductase (hDHFR). We then identified a compound by in silico screening that exploits this binding groove difference, and demonstrated that this compound inhibits CbDHFR with at least 25‐fold greater potency than hDHFR. Since this binding groove feature is shared by many other prokaryotes, the compound identified could form the basis of a novel antibacterial agent effective against a broad spectrum of pathogenic bacteria. Proteins 2015; 83:2124–2136. © 2015 Wiley Periodicals, Inc. 相似文献
10.
Przemyslaw Grudnik Dawid Debowski Anna Legowska Stanislaw Malicki Przemyslaw Golik Natalia Karna Krzysztof Rolka Grzegorz Dubin 《Proteins》2015,83(3):582-589
Protease inhibitors of the Bowman‐Birk (BBI) family are commonly found in plants and animals where they play a protective role against invading pathogens. Here, we report an atomic resolution (1Å) crystal structure of a peptide inhibitor isolated from a skin secretion of a Chinese bamboo odorous frog Huia versabilis (HV‐BBI) in complex with trypsin. HV‐BBI shares significant similarities in sequence with a previously described inhibitor from a diskless‐fingered odorous frog Odorrana graham (ORB). However, the latter is characterized by more than a 16,000 fold higher Ki against trypsin than HV‐BBI. Comparative analysis of trypsin cocrystal structures of HV‐BBI and ORB and additionally that of Sunflower Trypsin Inhibitor (SFTI‐1) together with accessory information on the affinities of inhibitor variants allowed us to pinpoint the inhibitor moiety responsible for the observed large difference in activity and also to define the extent of modifications permissible within the common protease‐binding loop scaffold of BBI inhibitors. We suggest that modifications outside of the inhibitory loop permit the evolution of specificity toward different enzymes characterized by trypsin‐like specificity. Proteins 2015; 83:582–589. © 2014 Wiley Periodicals, Inc. 相似文献
11.
The lifetime of the O intermediate of bacteriorhodopsin (BR) is extended by a factor of ～250 in the Leu93‐to‐Ala mutant (BR_L93A). To clarify the structural changes occurring in the last stage of the proton pumping cycle of BR, we crystallized BR_L93A into a hexagonal P622 crystal. Diffraction data from the unphotolyzed state showed that the deletion of three carbon atoms from Leu93 is compensated by the insertion of four water molecules in the cytoplasmic vicinity of retinal. This insertion of water is suggested to be responsible for the blue‐shifted λmax (540 nm) of the mutant. A long‐lived substate of O with a red‐shifted λmax (～565 nm) was trapped when the crystal of BR_L93A was flash‐cooled after illumination with green light. This substate (Oslow) bears considerable similarity to the M intermediate of native BR; that is, it commonly shows deformation of helix C and the FG loop, downward orientation of the side chain of Arg82, and disruption of the Glu194/Glu204 pair. In Oslow, however, the main chain of Lys216 is less distorted and retinal takes on the 13‐cis/15‐syn configuration. Another significant difference is seen in the pH dependence of the structure of the proton release group, the pKa value of which is suggested to be much lower in Oslow than in M. Proteins 2012;. © 2012 Wiley Periodicals, Inc. 相似文献
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14.
Michael C. Cavalier Zephan Melville Ehson Aligholizadeh E. Prabhu Raman Wenbo Yu Lei Fang Milad Alasady Adam D. Pierce Paul T. Wilder Alexander D. MacKerell David J. Weber 《Acta Crystallographica. Section D, Structural Biology》2016,72(6):753-760
Structure‐based drug discovery is under way to identify and develop small‐molecule S100B inhibitors (SBiXs). Such inhibitors have therapeutic potential for treating malignant melanoma, since high levels of S100B downregulate wild‐type p53 tumor suppressor function in this cancer. Computational and X‐ray crystallographic studies of two S100B–SBiX complexes are described, and both compounds (apomorphine hydrochloride and ethidium bromide) occupy an area of the S100B hydrophobic cleft which is termed site 3. These data also reveal novel protein–inhibitor interactions which can be used in future drug‐design studies to improve SBiX affinity and specificity. Of particular interest, apomorphine hydrochloride showed S100B‐dependent killing in melanoma cell assays, although the efficacy exceeds its affinity for S100B and implicates possible off‐target contributions. Because there are no structural data available for compounds occupying site 3 alone, these studies contribute towards the structure‐based approach to targeting S100B by including interactions with residues in site 3 of S100B. 相似文献
15.
Ji&#x;í Brynda Pavlína &#x;ez
ov Milan Fbry Magdalena Ho&#x;ej&#x;í Renata &#x;toura
ov Milan Sou
ek Martin Hradílek Jan Konvalinka Juraj Sedl
ek 《Acta Crystallographica. Section D, Structural Biology》2004,60(11):1943-1948
Depending on the excess of ligand used for complex formation, the HIV‐1 protease complexed with a novel phenylnorstatine inhibitor forms crystals of either hexagonal (P61) or orthorhombic (P212121) symmetry. The orthorhombic form shows an unusual complexity of crystal packing: in addition to one inhibitor molecule that is bound to the enzyme active site, the second inhibitor molecule is bound as an outer ligand at the protein interface. Binding of the outer ligand apparently increases the crystal‐quality parameters so that the diffraction data allow solution of the structure of the complex at 1.03 Å, the best resolution reported to date. The outer ligand interacts with all four surrounding HIV‐1 protease molecules and has a bent conformation owing to its accommodation in the intermolecular space. The parameters of the solved structures of the orthorhombic and hexagonal forms are compared. 相似文献
16.
Anna S. Gardberg Alexis Rae Del Castillo Kevin L. Weiss Flora Meilleur Matthew P. Blakeley Dean A. A. Myles 《Acta Crystallographica. Section D, Structural Biology》2010,66(5):558-567
The locations of H atoms in biological structures can be difficult to determine using X‐ray diffraction methods. Neutron diffraction offers a relatively greater scattering magnitude from H and D atoms. Here, 1.65 Å resolution neutron diffraction studies of fully perdeuterated and selectively CH3‐protonated perdeuterated crystals of Pyrococcus furiosus rubredoxin (D‐rubredoxin and HD‐rubredoxin, respectively) at room temperature (RT) are described, as well as 1.1 Å resolution X‐ray diffraction studies of the same protein at both RT and 100 K. The two techniques are quantitatively compared in terms of their power to directly provide atomic positions for D atoms and analyze the role played by atomic thermal motion by computing the σ level at the D‐atom coordinate in simulated‐annealing composite D‐OMIT maps. It is shown that 1.65 Å resolution RT neutron data for perdeuterated rubredoxin are ∼8 times more likely overall to provide high‐confidence positions for D atoms than 1.1 Å resolution X‐ray data at 100 K or RT. At or above the 1.0σ level, the joint X‐ray/neutron (XN) structures define 342/378 (90%) and 291/365 (80%) of the D‐atom positions for D‐rubredoxin and HD‐rubredoxin, respectively. The X‐ray‐only 1.1 Å resolution 100 K structures determine only 19/388 (5%) and 8/388 (2%) of the D‐atom positions above the 1.0σ level for D‐rubredoxin and HD‐rubredoxin, respectively. Furthermore, the improved model obtained from joint XN refinement yielded improved electron‐density maps, permitting the location of more D atoms than electron‐density maps from models refined against X‐ray data only. 相似文献
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18.
Zhao M Cascio D Sawaya MR Eisenberg D 《Protein science : a publication of the Protein Society》2011,20(6):996-1004
Aggregates of the protein α-synuclein are the main component of Lewy bodies, the hallmark of Parkinson's disease. α-Synuclein aggregates are also found in many human neurodegenerative diseases known as synucleinopathies. In vivo, α-synuclein associates with membranes and adopts α-helical conformations. The details of how α-synuclein converts from the functional native state to amyloid aggregates remain unknown. In this study, we use maltose-binding protein (MBP) as a carrier to crystallize segments of α-synuclein. From crystal structures of fusions between MBP and four segments of α-synuclein, we have been able to trace a virtual model of the first 72 residues of α-synuclein. Instead of a mostly α-helical conformation observed in the lipid environment, our crystal structures show α-helices only at residues 1-13 and 20-34. The remaining segments are extended loops or coils. All of the predicted fiber-forming segments based on the 3D profile method are in extended conformations. We further show that the MBP fusion proteins with fiber-forming segments from α-synuclein can also form fiber-like nano-crystals or amyloid-like fibrils. Our structures suggest intermediate states during amyloid formation of α-synuclein. 相似文献
19.
In a crystallography experiment, a crystal is irradiated with X-rays whose diffracted waves are collected and measured. The reconstruction of the structure of the molecule in the crystal requires knowledge of the phase of the diffracted waves, information that is lost in the passage from the three-dimensional structure of the molecule to its diffraction pattern. It can be recovered using experimental methods such as heavy-atom isomorphous replacement and anomalous scattering or by molecular replacement, which relies on the availability of an atomic model of the target structure. This can be the structure of the target protein itself, if a previous structure determination is available, or a computational model or, in some cases, the structure of a homologous protein. It is not straightforward to predict beforehand whether or not a computational model will work in a molecular replacement experiment, although some rules of thumb exist. The consensus is that even minor differences in the quality of the model, which are rather difficult to estimate a priori, can have a significant effect on the outcome of the procedure. We describe here a method for quickly assessing whether a protein structure can be solved by molecular replacement. The procedure consists in submitting the sequence of the target protein to a selected list of freely available structure prediction servers, cluster the resulting models, select the representative structures of each cluster and use them as search models in an automatic phasing procedure. We tested the procedure using the structure factors of newly released proteins of known structure downloaded from the Protein Data Bank as soon as they were made available. Using our automatic procedure we were able to obtain an interpretable electron density map in more than half the cases. 相似文献
20.
El-Kabbani O Darmanin C Schneider TR Hazemann I Ruiz F Oka M Joachimiak A Schulze-Briese C Tomizaki T Mitschler A Podjarny A 《Proteins》2004,55(4):805-813
The X-ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK-860) and Minalrestat (WAY-509) were determined at atomic resolutions of 0.92 A and 1.1 A, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion-binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestat's hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group inside the \"specificity\" pocket. The interactions between Minalrestat's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main-chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active-site residue His110. The 1'-position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nepsilon2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H-atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl-ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design. 相似文献