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At least 1,243 records · Page 69Linked to original sources
PubMed2000

Solution structure of the smallest cofactor-active fragment of thrombomodulin.

A glycosylated fragment of thrombomodulin containing two epidermal growth factor-like domains (TMEGF45) was analyzed by NMR. The 4th-domains structure of this two-domain fragment is similar to that of the individual domain previously determined. The 5th-domain, which has uncrossed disulfide bonds, is not as well determined in the two-domain fragment than the individual domain previously solved. The flexibility of the 5th-domain is consistent with low heteronuclear NOEs. In the individual 5th-domain, Met 388 was disordered, and key thrombin binding residues formed a hydrophobic core. By contrast, in TMEGF45, Met 388 is in the 5th-domain core, positioned by Phe 376 from the 4th-domain. As a result, key thrombin binding residues that were in the core of the individual domain are expelled. Upon thrombin binding, chemical shifts of two residues in the 4th-domain, the three interdomain linker residues, and nearly all of the 5th-domain are perturbed. Thus, TMEGF45 binds thrombin by an induced fit mechanism involving a flexible 5th-domain.

Acetylglucosamine↗
PubMed2000

Substrate-induced exposure of an energy-coupling motif of a membrane transporter.

BtuB is an outer membrane protein responsible for the uptake of vitamin B12 by Escherichia coli. It belongs to a family of bacterial transport proteins that derive energy for transport by coupling to the trans-periplasmic energy-coupling protein TonB. Using site-directed spin labeling and EPR we investigated the structure and substrate-induced changes in the TonB box, a highly conserved region in all TonB dependent transporters that may couple to TonB. In the absence of substrate, the line widths and collision parameters from EPR are consistent with this domain existing in a structured helical conformation that contacts the barrel of the transporter. Addition of substrate converts this segment into an extended structure that is highly dynamic, disordered and probably extended into the periplasm. This structural change demonstrates that the TonB box cycles between sequestered and accessible states in a substrate-dependent fashion. In a transport defective mutant of BtuB, this conformational cycle is disrupted and the TonB box appears to be extended even in the absence of substrate. These data suggest that the TonB box extends into the periplasm and interacts with TonB only in

Amino Acid Motifs↗
PubMed2000

Site-site communication in the EF-hand Ca2+-binding protein calbindin D9k.

The cooperative binding of Ca2+ ions is an essential functional property of the EF-hand family of Ca2+-binding proteins. To understand how these proteins function, it is essential to characterize intermediate binding states in addition to the apo- and holo-proteins. The three-dimensional solution structure and fast time scale internal motional dynamics of the backbone have been determined for the half-saturated state of the N56A mutant of calbindin D9k with Ca2+ bound only in the N-terminal site. The extent of conformational reorganization and a loss of flexibility in the C-terminal EF-hand upon binding of an ion in the N-terminal EF-hand provide clear evidence of the importance of site-site interactions in this family of proteins, and demonstrates the strength of long-range effects in the cooperative EF-hand Ca2+-binding domain.

Allosteric Site↗
PubMed2000

Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2.

Leech carboxypeptidase inhibitor (LCI) is a novel protein inhibitor present in the medicinal leech Hirudo medicinalis. The structures of LCI free and bound to carboxypeptidase A2 (CPA2)have been determined by NMR and X-ray crystallography, respectively. The LCI structure defines a new protein motif that comprises a five-stranded antiparallel beta-sheet and one short alpha-helix. This structure is preserved in the complex with human CPA2 in the X-ray structure, where the contact regions between the inhibitor and the protease are defined. The C-terminal tail of LCI becomes rigid upon binding the protease as shown in the NMR relaxation studies, and it interacts with the carboxypeptidase in a substrate-like manner. The homology between the C-terminal tails of LCI and the potato carboxypeptidase inhibitor represents a striking example of convergent evolution dictated by the target protease. These new structures are of biotechnological interest since they could elucidate the control mechanism of metallo-carboxypeptidases and could be used as lead compounds for the search of fibrinolytic drugs.

Amino Acid Sequence↗
PubMed2000

Crystal structure of the bacterial conjugation repressor finO.

The conjugative transfer of F-like plasmids is repressed by FinO, an RNA binding protein. FinO interacts with the F-plasmid encoded traJ mRNA and its antisense RNA, FinP, stabilizing FinP against endonucleolytic degradation and facilitating sense-antisense RNA recognition. Here we present the 2.0 A resolution X-ray crystal structure of FinO, lacking its flexible N-terminal extension. FinO adopts a novel, elongated, largely helical conformation. An N-terminal region, previously shown to contact RNA, forms a positively charged alpha-helix (helix 1) that protrudes 45 A from the central core of FinO. A C-terminal region of FinO that is implicated in RNA interactions also extends out from the central body of the protein, adopting a helical conformation and packing against the base of the N-terminal helix. A highly positively charged patch on the surface of the FinO core may present another RNA binding surface. The results of an in vitro RNA duplexing assay demonstrate that the flexible N-terminal region of FinO plays a key role in FinP-traJ RNA recognition, and supports our proposal that this region and the N-terminus of helix 1 interact with and stabilize paired, complementary RNA loops in a kissing complex.

Amino Acid Sequence↗
PubMed2000

Protein dynamics from NMR.

This review surveys recent investigations of conformational fluctuations of proteins in solution using NMR techniques. Advances in experimental methods have provided more accurate means of characterizing fast and slow internal motions as well as overall diffusion. The information obtained from NMR dynamics experiments provides insights into specific structural changes or configurational energetics associated with function. A variety of applications illustrate that studies of protein dynamics provide insights into protein-protein interactions, target recognition, ligand binding, and enzyme function.

Amides↗
PubMed2000

Evidence for cleft closure in actomyosin upon ADP release.

Structural insights into the interaction of smooth muscle myosin with actin have been provided by computer-based fitting of crystal structures into three-dimensional reconstructions obtained by electron cryomicroscopy, and by mapping of structural and dynamic changes in the actomyosin complex. The actomyosin structures determined in the presence and absence of MgADP differ significantly from each other, and from all crystallographic structures of unbound myosin. Coupled to a complex movement ( approximately 34 A) of the light chain binding domain upon MgADP release, we observed a approximately 9 degrees rotation of the myosin motor domain relative to the actin filament, and a closure of the cleft that divides the actin binding region of the myosin head. Cleft closure is achieved by a movement of the upper 50 kDa region, while parts of the lower 50 kDa region are stabilized through strong interactions with actin. This model supports a mechanism in which binding of MgATP at the active site opens the cleft and disrupts the interface, thereby releasing myosin from actin.

Actins↗
PubMed2000

Crystal structure of the bacterial protein export chaperone secB.

SecB is a bacterial molecular chaperone involved in mediating translocation of newly synthesized polypeptides across the cytoplasmic membrane of bacteria. The crystal structure of SecB from Haemophilus influenzae shows that the molecule is a tetramer organized as a dimer of dimers. Two long channels run along the side of the molecule. These are bounded by flexible loops and lined with conserved hydrophobic amino acids, which define a suitable environment for binding non-native polypeptides. The structure also reveals an acidic region on the top surface of the molecule, several residues of which have been implicated in binding to SecA, its downstream target.

Adenosine Triphosphatases↗
PubMed2001

The neuronal t-SNARE complex is a parallel four-helix bundle.

Assembly of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex is an essential step for neurotransmitter release in synapses. The presynaptic plasma membrane associated proteins (t-SNAREs), SNAP-25 (synaptosome-associated protein of 25,000 Da) and syntaxin 1A may form an intermediate complex that later binds to vesicle-associated membrane protein 2 (VAMP2). Using spin labeling electron paramagnetic resonance (EPR), we found that the two t-SNARE proteins assemble into a parallel four-helix bundle that consists of two identical syntaxin 1A components and the N-terminal and C-terminal domains of SNAP-25. Although the structure is generally similar to that of the final SNARE complex, the middle region of the helical bundle appears more flexible in the t-SNARE complex. Such flexibility might facilitate interactions between VAMP2 and the t-SNARE complex.

Electron Spin Resonance Spectroscopy↗
PubMed2001

ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy.

Kinesin is an ATP-driven molecular motor protein that moves processively along microtubules. Despite considerable research, the detailed mechanism of kinesin motion remains elusive. We applied an enhanced suite of single- and multiple-molecule fluorescence polarization microscopy assays to report the orientation and mobility of kinesin molecules bound to microtubules as a function of nucleotide state. In the presence of analogs of ATP, ADP-Pi or in the absence of nucleotide, the kinesin head maintains a rigid orientation. In the presence of ADP, the motor domain of kinesin, still bound to the microtubule, adopts a previously undescribed, highly mobile state. This state may be general to the chemomechanical cycle of motor proteins; in the case of kinesin, the transition from a highly mobile to a rigid state after ADP release may contribute to the generation of the 8 nm step.

Adenosine Diphosphate↗
PubMed2003

Structure of the core domain of human cardiac troponin in the Ca(2+)-saturated form.

Troponin is essential in Ca(2+) regulation of skeletal and cardiac muscle contraction. It consists of three subunits (TnT, TnC and TnI) and, together with tropomyosin, is located on the actin filament. Here we present crystal structures of the core domains (relative molecular mass of 46,000 and 52,000) of human cardiac troponin in the Ca(2+)-saturated form. Analysis of the four-molecule structures reveals that the core domain is further divided into structurally distinct subdomains that are connected by flexible linkers, making the entire molecule highly flexible. The alpha-helical coiled-coil formed between TnT and TnI is integrated in a rigid and asymmetric structure (about 80 angstrom long), the IT arm, which bridges putative tropomyosin-anchoring regions. The structures of the troponin ternary complex imply that Ca(2+) binding to the regulatory site of TnC removes the carboxy-terminal portion of TnI from actin, thereby altering the mobility and/or flexibility of troponin and tropomyosin on the actin filament.

Calcium↗
PubMed2003

Visualization of an unstable coiled coil from the scallop myosin rod.

Alpha-helical coiled coils in muscle exemplify simplicity and economy of protein design: small variations in sequence lead to remarkable diversity in cellular functions. Myosin II is the key protein in muscle contraction, and the molecule's two-chain alpha-helical coiled-coil rod region--towards the carboxy terminus of the heavy chain--has unusual structural and dynamic features. The amino-terminal subfragment-2 (S2) domains of the rods can swing out from the thick filament backbone at a hinge in the coiled coil, allowing the two myosin 'heads' and their motor domains to interact with actin and generate tension. Most of the S2 rod appears to be a flexible coiled coil, but studies suggest that the structure at the N-terminal region is unstable, and unwinding or bending of the alpha-helices near the head-rod junction seems necessary for many of myosin's functional properties. Here we show the physical basis of a particularly weak coiled-coil segment by determining the 2.5-A-resolution crystal structure of a leucine-zipper-stabilized fragment of the scallop striated-muscle myosin rod adjacent to the head-rod junction. The N-terminal 14 residues are poorly ordered; the rest of the S2 segment forms a flexible coiled coil with poorly packed core residues. The unusual absence of interhelical salt bridges here exposes apolar core atoms to solvent.

Amino Acid Sequence↗
PubMed2004

Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism.

The bacterial flagellum is a motile organelle, and the flagellar hook is a short, highly curved tubular structure that connects the flagellar motor to the long filament acting as a helical propeller. The hook is made of about 120 copies of a single protein, FlgE, and its function as a nano-sized universal joint is essential for dynamic and efficient bacterial motility and taxis. It transmits the motor torque to the helical propeller over a wide range of its orientation for swimming and tumbling. Here we report a partial atomic model of the hook obtained by X-ray crystallography of FlgE31, a major proteolytic fragment of FlgE lacking unfolded terminal regions, and by electron cryomicroscopy and three-dimensional helical image reconstruction of the hook. The model reveals the intricate molecular interactions and a plausible switching mechanism for the hook to be flexible in bending but rigid against twisting for its universal joint function.

Bacterial Proteins↗
PubMed2005

Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly.

The atomic structure of tubulin in a polymerized, straight protofilament is clearly distinct from that in a curved conformation bound to a cellular depolymerizer. The nucleotide contents are identical, and in both cases the conformation of the GTP-containing, intra-dimer interface is indistinguishable from the GDP-containing, inter-dimer contact. Here we present two structures corresponding to the start and end points in the microtubule polymerization and hydrolysis cycles that illustrate the consequences of nucleotide state on longitudinal and lateral assembly. In the absence of depolymerizers, GDP-bound tubulin shows distinctive intra-dimer and inter-dimer interactions and thus distinguishes the GTP and GDP interfaces. A cold-stable tubulin polymer with the non-hydrolysable GTP analogue GMPCPP, containing semi-conserved lateral interactions, supports a model in which the straightening of longitudinal interfaces happens sequentially, starting with a conformational change after GTP binding that straightens the dimer enough for the formation of lateral contacts into a non-tubular intermediate. Closure into a microtubule does not require GTP hydrolysis.

Cryoelectron Microscopy↗
PubMed2005

A structural basis for allosteric control of DNA recombination by lambda integrase.

Site-specific DNA recombination is important for basic cellular functions including viral integration, control of gene expression, production of genetic diversity and segregation of newly replicated chromosomes, and is used by bacteriophage lambda to integrate or excise its genome into and out of the host chromosome. lambda recombination is carried out by the bacteriophage-encoded integrase protein (lambda-int) together with accessory DNA sites and associated bending proteins that allow regulation in response to cell physiology. Here we report the crystal structures of lambda-int in higher-order complexes with substrates and regulatory DNAs representing different intermediates along the reaction pathway. The structures show how the simultaneous binding of two separate domains of lambda-int to DNA facilitates synapsis and can specify the order of DNA strand cleavage and exchange. An intertwined layer of amino-terminal domains bound to accessory (arm) DNAs shapes the recombination complex in a way that suggests how arm binding shifts the reaction equilibrium in favour of recombinant products.

Allosteric Regulation↗
PubMed2005

Structural mechanism for sterol sensing and transport by OSBP-related proteins.

The oxysterol-binding-protein (OSBP)-related proteins (ORPs) are conserved from yeast to humans, and are implicated in the regulation of sterol homeostasis and in signal transduction pathways. Here we report the structure of the full-length yeast ORP Osh4 (also known as Kes1) at 1.5-1.9 A resolution in complexes with ergosterol, cholesterol, and 7-, 20- and 25-hydroxycholesterol. We find that a single sterol molecule binds within a hydrophobic tunnel in a manner consistent with a transport function for ORPs. The entrance is blocked by a flexible amino-terminal lid and surrounded by basic residues that are critical for Osh4 function. The structure of the open state of a lid-truncated form of Osh4 was determined at 2.5 A resolution. Structural analysis and limited proteolysis show that sterol binding closes the lid and stabilizes a conformation favouring transport across aqueous barriers and signal transmission. The structure of Osh4 in the absence of ligand exposes potential phospholipid-binding sites that are positioned for membrane docking and sterol exchange. On the basis of these observations, we propose a model in which sterol and membrane binding promote reciprocal conformational changes that facilitate a sterol transfer and signalling cycle.

Biological Transport↗