Crystal structure of monomeric native antithrombin reveals a novel reactive center loop conformation

The poor inhibitory activity of circulating antithrombin (AT) is critical to the formation of blood clots at sites of vascular damage. AT becomes an efficient inhibitor of the coagulation proteases only after binding to a specific heparin pentasaccharide, which alters the conformation of the reactive center loop (RCL). The molecular basis of this activation event lies at the heart of the regulation of hemostasis and accounts for the anticoagulant properties of the low molecular weight heparins. Although several structures of AT have been solved, the conformation of the RCL in native AT remains unknown because of the obligate crystal contact between the RCL of native AT and its latent counterpart. Here we report the crystallographic structure of a variant of AT in its monomeric native state. The RCL shifted approximately 20 A, and a salt bridge was observed between the P1 residue (Arg-393) and Glu-237. This contact explains the effect of mutations at the P1 position on the affinity of AT for heparin and also the properties of AT-Truro (E237K). The relevance of the observed conformation was verified through mutagenesis studies and by solving structures of the same variant in different crystal forms. We conclude that the poor inhibitory activity of the circulating form of AT is partially conferred by intramolecular contacts that restrain the RCL, orient the P1 residue away from attacking proteases, and additionally block the exosite utilized in protease recognition.     

Crystal structure of the mitotic spindle kinesin Eg5 reveals a novel conformation of the neck-linker

Success of mitosis depends upon the coordinated and regulated activity of many cellular factors, including kinesin motor proteins, which are required for the assembly and function of the mitotic spindle. Eg5 is a kinesin implicated in the formation of the bipolar spindle and its movement prior to and during anaphase. We have determined the crystal structure of the Eg5 motor domain with ADP-Mg bound. This structure revealed a new intramolecular binding site of the neck-linker. In other kinesins, the neck-linker has been shown to be a critical mechanical element for force generation. The neck-linker of conventional kinesin is believed to undergo an ordered-to-disordered transition as it translocates along a microtubule. The structure of Eg5 showed an ordered neck-linker conformation in a position never observed previously. The docking of the neck-linker relies upon residues conserved only in the Eg5 subfamily of kinesin motors. Based on this new information, we suggest that the neck-linker of Eg5 may undergo an ordered-to-ordered transition during force production. This ratchet-like mechanism is consistent with the biological activity of Eg5.     

The role of metal ions in the conformation of the four-way DNA junction.

Metal ions fold DNA junctions into a compact conformation that confers protection of all thymine bases to modification by osmium tetroxide. In the absence of the cation the arms of the junction are fully extended in an approximately square-planar configuration. Group IIa cations are effective in achieving a folded conformation of the junction at 80-100 microM, and there is an excellent agreement between the ionic concentrations that fold the junctions as deduced from gel electrophoretic experiments, and those that prevent osmium tetroxide reaction at the junction. Hexamminecobalt(III) achieves full folding at 2 microM, while spermine and spermidine are effective at 25 microM. Some transition metal ions such as Ni(II) may replace the group IIA cations. Monovalent ions of group IA are only partially effective in folding the junctions. Very much higher concentrations are necessary, gel electrophoretic mobilities suggest that a less symmetrical conformation is adopted and thymine bases at the junction remain reactive to osmium tetroxide. Charge-charge interactions at the centre of the junction are structurally extremely important. Substitution of junction phosphate groups by uncharged methyl phosphonates severely perturbs the structure of the junction. If just two phosphates are substituted, diametrically facing across the junction, the structure always folds in order to place the electrically neutral phosphate on the exchanging strands. We suggest that folding of the junction into the stacked X-structure generates electronegative clefts that can selectively bind metal ions, depending on the chemistry, size and charge of the ion. Moreover, occupation of these cavities is essential for junction folding, in order to reduce electrostatic repulsion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of the N-terminal ankyrin repeat domain of TRPV3 reveals unique conformation of finger 3 loop critical for channel function

In all six members of TRPV channel subfamily, there is an ankyrin repeat domain (ARD) in their intracellular Ntermini. Ankyrin (ANK) repeat, a common motif with typically 33 residues in each repeat, is primarily involved in protein-protein interactions. Despite the sequence similarity among the ARDs of TRPV channels, the structure of TRPV3-ARD, however, remains unknown. Here, we report the crystal structure of TRPV3-ARD solved at 1.95 ? resolution, which reveals six-ankyrin repeats. While overall structure of TRPV3-ARD is similar to ARDs from other members of TRPV subfamily; it, however, features a noticeable finger 3 loop that bends over and is stabilized by a network of hydrogen bonds and hydrophobic packing, instead of being flexible as seen in known TRPV-ARD structures. Electrophysiological recordings demonstrated that mutating key residues R225, R226, Q255, and F249 of finger 3 loop altered the channel activities and pharmacology. Taken all together, our findings show that TRPV3-ARD with characteristic finger 3 loop likely plays an important role in channel function and pharmacology.     

Crystal structure of thrombin in a self-inhibited conformation

The activating effect of Na(+) on thrombin is allosteric and depends on the conformational transition from a low activity Na(+)-free (slow) form to a high activity Na(+)-bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na(+) have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na(+) site and the active site regions. Here, we report a 1.87A resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow --> fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10A to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na(+)-binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na(+) and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215.     

Solution structure of the lymphocyte receptor Nkrp1a reveals a distinct conformation of the long loop region as compared to in the crystal structure

Mouse Nkrp1a receptor is a C‐type lectin‐like receptor expressed on the surface of natural killer cells that play an important role against virally infected and tumor cells. The recently solved crystal structure of Nkrp1a raises questions about a long loop region which was uniquely extended from the central region in the crystal. To understand the functional significance of the loop, the solution structure of Nkrp1a using nuclear magnetic resonance (NMR) spectroscopy was determined. A notable difference between the crystal and NMR structure of Nkrp1a appears in the conformation of the long loop region. While the extended loop points away from the central core and mediates formation of a domain swapped dimer in the crystal, the solution structure is monomeric with the loop tightly anchored to the central region. The findings described the first solution structure in the Nkrp1 family and revealed intriguing similarities and differences to the crystal structure. Proteins 2016; 84:1304–1311. © 2016 Wiley Periodicals, Inc.     

Crystal structure of a micro-like calpain reveals a partially activated conformation with low Ca2+ requirement

The two Ca2+-dependent cysteine proteases, micro- and m-calpain, are involved in various Ca2+-linked signal pathways but differ markedly in their Ca2+ requirements for activation. We have determined the structure of a micro-like calpain, which has 85% micro-calpain sequence (the first 48 and the last 62 residues of the large subunit are those from m-calpain) and a low Ca2+ requirement. This construct was used because micro-calpain itself is too poorly expressed. The structure of micro-like calpain is very similar in overall fold to that of m-calpain as expected, but differs significantly in two aspects. In comparison with m-calpain, the catalytic triad residues in micro-like calpain, His and Cys, are much closer together in the absence of Ca2+, and significant portions of the Ca2+ binding EF-hand motifs are disordered and more flexible. These structural differences imply that Ca2+-free micro-calpain may represent a partially activated structure, requiring lower Ca2+ concentration to trigger its activation.     

Crystal structure of the A domain from complement factor B reveals an integrin-like open conformation

Complement factor B is a 90 kDa protein consisting of three domains: a three-module complement control protein, a von Willebrand factor A domain, and a C-terminal serine protease (SP) domain that adopts a default inactive (zymogen) conformation. The interaction between factor B and pathogen-bound C3b is mediated by its A domain, triggering a conformational change in factor B that ultimately creates the "C3 convertase" of the alternative complement pathway. We report the crystal structure of the A domain from factor B and show that it contains an integrin-like MIDAS motif that adopts the "open" conformation typical of integrin-ligand complexes, with an acidic residue (provided by a fortuitous crystal contact) completing the coordination of the metal ion. Modeling studies indicate that the factor B A domain can also adopt the closed conformation, supporting the hypothesis that an "integrin-like switch" is conserved in complement proteins and perhaps in 60 other A domains found within the human proteome.     

Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules

Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.     

Crystal structure of agmatinase reveals structural conservation and inhibition mechanism of the ureohydrolase superfamily

Agmatine is the product of arginine decarboxylation and can be hydrolyzed by agmatinase to putrescine, the precursor for biosynthesis of higher polyamines, spermidine, and spermine. Besides being an intermediate in polyamine metabolism, recent findings indicate that agmatine may play important regulatory roles in mammals. Agmatinase is a binuclear manganese metalloenzyme and belongs to the ureohydrolase superfamily that includes arginase, formiminoglutamase, and proclavaminate amidinohydrolase. Compared with a wealth of structural information available for arginases, no three-dimensional structure of agmatinase has been reported. Agmatinase from Deinococcus radiodurans, a 304-residue protein, shows approximately 33% of sequence identity to human mitochondrial agmatinase. Here we report the crystal structure of D. radiodurans agmatinase in Mn(2+)-free, Mn(2+)-bound, and Mn(2+)-inhibitor-bound forms, representing the first structure of agmatinase. It reveals the conservation as well as variation in folding, oligomerization, and the active site of the ureohydrolase superfamily. D. radiodurans agmatinase exists as a compact homohexamer of 32 symmetry. Its binuclear manganese cluster is highly similar but not identical to the clusters of arginase and proclavaminate amidinohydrolase. The structure of the inhibited complex reveals that inhibition by 1,6-diaminohexane arises from the displacement of the metal-bridging water.     

Crystal structure of M-Ras reveals a GTP-bound "off" state conformation of Ras family small GTPases

Although some members of Ras family small GTPases, including M-Ras, share the primary structure of their effector regions with Ras, they exhibit vastly different binding properties to Ras effectors such as c-Raf-1. We have solved the crystal structure of M-Ras in the GDP-bound and guanosine 5'-(beta,gamma-imido)triphosphate (Gpp(NH)p)-bound forms. The overall structure of M-Ras resembles those of H-Ras and Rap2A, except that M-Ras-Gpp(NH)p exhibits a distinctive switch I conformation, which is caused by impaired intramolecular interactions between Thr-45 (corresponding to Thr-35 of H-Ras) of the effector region and the gamma-phosphate of Gpp(NH)p. Previous 31P NMR studies showed that H-Ras-Gpp(NH)p exists in two interconverting conformations, states 1 and 2. Whereas state 2 is a predominant form of H-Ras and corresponds to the "on" conformation found in the complex with effectors, state 1 is thought to represent the "off" conformation, whose tertiary structure remains unknown. 31P NMR analysis shows that free M-Ras-Gpp(NH)p predominantly assumes the state 1 conformation, which undergoes conformational transition to state 2 upon association with c-Raf-1. These results indicate that the solved structure of M-Ras-Gp-p(NH)p corresponds to the state 1 conformation. The predominance of state 1 in M-Ras is likely to account for its weak binding ability to the Ras effectors, suggesting the importance of the tertiary structure factor in small GTPase-effector interaction. Further, the first determination of the state 1 structure provides a molecular basis for developing novel anti-cancer drugs as compounds that hold Ras in the state 1 "off" conformation.     

A CD study of the role of metal ions in the conformation of poly(L-lysine)

The effect of LiCl, NaCl, and CsCl as univalent salts, and of CaCl₂, ZnCl₂, and MgCl₂ as divalent salts, on the α and antiparallel β-sheet, and random conformations of poly(L-lysine) (PLL), in water at room temperature were examined by means of CD and compared quantitatively on the basis of elliptical strength at the maximal peak. Changes in the α-helical and antiparallel β-sheet helical conformations of PLL were markedly dependent on the salt concentrations of LiCl, NaCl, and CsCl, which induced decreases in negative intensity in that order. The CD spectrum of the random conformation, the most disordered form, displayed positive cotton effect in concentrations of these salts up to 3.0M and a negative peak in concentrations of 6.0M. The effect of these salts on the random conformation of PLL was stronger than that on the α- and β-conformations in higher concentrations. The CD spectrum of the random conformation in the presence of CaCl₂, ZnCl₂, and MgCl₂, on the other hand, showed negative cotton effect in salt concentrations as low as 3.0M. It was impossible, however, to measure the effect on α- and β-conformations of ZnCl₂ and MgCl₂ above concentrations of 10 mM because of a solubility problem with salts in alkaline solution.     

Crystal structure of baculovirus P35 reveals a novel conformational change in the reactive site loop after caspase cleavage

Baculovirus P35 is a universal suppressor of apoptosis that stoichiometrically inhibits cellular caspases in a novel cleavage-dependent mechanism. Upon caspase cleavage at Asp-87, the 10- and 25-kDa cleavage products of P35 remain tightly associated with the inhibited caspase. Mutations in the alpha-helix of the reactive site loop preceding the cleavage site abrogate caspase inhibition and antiapoptotic activity. Substitution of Pro for Val-71, which is located in the middle of this alpha-helix, produces a protein that is cleaved at the requisite Asp-87 but does not remain bound to the caspase. This loss-of-function mutation provided the opportunity to structurally analyze the conformational changes of the P35 reactive site loop after caspase cleavage. We report here the 2.7 A resolution crystal structure of V71P-mutated P35 after cleavage by human caspase-3. The structure reveals a large movement in the carboxyl-terminal side of the reactive site loop that swings down and forms a new beta-strand that augments an existing beta-sheet. Additionally, the hydrophobic amino terminus releases and extends away from the protein core. Similar movements occur when P35 forms an inhibitory complex with human caspase-8. These findings suggest that the alpha-helix mutation may alter the sequential steps or kinetics of the conformational changes required for inhibition, thereby causing P35 loss of function.     

A crystal structure of the human protein kinase Mps1 reveals an ordered conformation of the activation loop

Monopolar spindle 1 (Mps1) is a dual-specificity protein kinase, orchestrating faithful chromosome segregation during mitosis. All reported structures of the Mps1 kinase adopt the hallmarks of an inactive conformation, which includes a mostly disordered activation loop. Here, we present a 2.4 Å resolution crystal structure of an “extended” version of the Mps1 kinase domain, which shows an ordered activation loop. However, the other structural characteristics of an active kinase are not present. Our structure shows that the Mps1 activation loop can fit to the ATP binding pocket and interferes with ATP, but less so with inhibitors binding, partly explain the potency of various Mps1 inhibitors.     

Crystal structure of the beta-apical domain of the thermosome reveals structural plasticity in the protrusion region

The crystal structure of the beta-apical domain of the thermosome, an archaeal group II chaperonin from Thermoplasma acidophilum, has been determined at 2.8 A resolution. The structure shows an invariant globular core from which a 25 A long protrusion emanates, composed of an elongated alpha-helix (H10) and a long extended stretch consisting of residues GluB245-ThrB253. A comparison with previous apical domain structures reveals a large segmental displacement of the protruding part of helix H10 via the hinge GluB276-ValB278. The region comprising residues GluB245-ThrB253 adopts an extended beta-like conformation rather than the alpha-helix seen in the alpha-apical domain. Consequently, it appears that the protrusions of the apical domains from group II chaperonins might assume a variety of context-dependent conformations during an open, substrate-accepting state of the chaperonin. Sequence variations in the protrusion regions that are found in the eukaryotic TRiC/CCT subunits may provide different structural propensities and hence serve different roles in substrate recognition.     

Crystal structure of a mono- and diacylglycerol lipase from Malassezia globosa reveals a novel lid conformation and insights into the substrate specificity

Most lipases contain a lid domain to shield the hydrophobic binding site from the water environment. The lid, mostly in helical form, can undergo a conformational change to expose the active cleft during the interfacial activation. Here we report the crystal structures of Malassezia globosa LIP1 (SMG1) at 1.45 and 2.60 ? resolution in two crystal forms. The structures present SMG1 in its closed form, with a novel lid in loop conformation. SMG1 is one of the few members in the fungal lipase family that has been found to be strictly specific for mono- and diacylglycerol. To date, the mechanism for this substrate specificity remains largely unknown. To investigate the substrate binding properties, we built a model of SMG1 in open conformation. Based on this model, we found that the two bulky hydrophobic residues adjacent to the catalytic site and the N-terminal hinge region of the lid both may act as steric hindrances for triacylglycerols binding. These unique structural features of SMG1 will provide a better understanding on the substrate specificity of mono- and diacylglycerol lipases and a platform for further functional study of this enzyme.