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At least 1,261 records · Page 70Linked to original sources
PubMed2005

Mesoscale conformational changes in the DNA-repair complex Rad50/Mre11/Nbs1 upon binding DNA.

The human Rad50/Mre11/Nbs1 complex (hR/M/N) functions as an essential guardian of genome integrity by directing the proper processing of DNA ends, including DNA breaks. This biological function results from its ability to tether broken DNA molecules. hR/M/N's dynamic molecular architecture consists of a globular DNA-binding domain from which two 50-nm-long coiled coils protrude. The coiled coils are flexible and their apices can self-associate. The flexibility of the coiled coils allows their apices to adopt an orientation favourable for interaction. However, this also allows interaction between the tips of two coiled coils within the same complex, which competes with and frustrates the intercomplex interaction required for DNA tethering. Here we show that the dynamic architecture of hR/M/N is markedly affected by DNA binding. DNA binding by the hR/M/N globular domain leads to parallel orientation of the coiled coils; this prevents intracomplex interactions and favours intercomplex associations needed for DNA tethering. The hR/M/N complex thus is an example of a biological nanomachine in which binding to its ligand, in this case DNA, affects the functional conformation of a domain located 50 nm distant.

Acid Anhydride Hydrolases↗
PubMed2005

Microscopic artificial swimmers.

Microorganisms such as bacteria and many eukaryotic cells propel themselves with hair-like structures known as flagella, which can exhibit a variety of structures and movement patterns. For example, bacterial flagella are helically shaped and driven at their bases by a reversible rotary engine, which rotates the attached flagellum to give a motion similar to that of a corkscrew. In contrast, eukaryotic cells use flagella that resemble elastic rods and exhibit a beating motion: internally generated stresses give rise to a series of bends that propagate towards the tip. In contrast to this variety of swimming strategies encountered in nature, a controlled swimming motion of artificial micrometre-sized structures has not yet been realized. Here we show that a linear chain of colloidal magnetic particles linked by DNA and attached to a red blood cell can act as a flexible artificial flagellum. The filament aligns with an external uniform magnetic field and is readily actuated by oscillating a transverse field. We find that the actuation induces a beating pattern that propels the structure, and that the external fields can be adjusted to control the velocity and the direction of motion.

Biomimetic Materials↗
PubMed2005

Intrinsic dynamics of an enzyme underlies catalysis.

A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis. The concept of conformational substates has been described; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis-trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.

Binding Sites↗
PubMed2005

Structure of the E. coli protein-conducting channel bound to a translating ribosome.

Secreted and membrane proteins are translocated across or into cell membranes through a protein-conducting channel (PCC). Here we present a cryo-electron microscopy reconstruction of the Escherichia coli PCC, SecYEG, complexed with the ribosome and a nascent chain containing a signal anchor. This reconstruction shows a messenger RNA, three transfer RNAs, the nascent chain, and detailed features of both a translocating PCC and a second, non-translocating PCC bound to mRNA hairpins. The translocating PCC forms connections with ribosomal RNA hairpins on two sides and ribosomal proteins at the back, leaving a frontal opening. Normal mode-based flexible fitting of the archaeal SecYEbeta structure into the PCC electron microscopy densities favours a front-to-front arrangement of two SecYEG complexes in the PCC, and supports channel formation by the opening of two linked SecY halves during polypeptide translocation. On the basis of our observation in the translocating PCC of two segregated pores with different degrees of access to bulk lipid, we propose a model for co-translational protein translocation.

Cell Membrane↗
PubMed2005

Structures of ParB bound to DNA reveal mechanism of partition complex formation.

The faithful inheritance of genetic information, which is essential for all organisms, requires accurate DNA partition (segregation) at cell division. In prokaryotes, partition is mediated by par systems, for which the P1 plasmid system of Escherichia coli is a prototype comprising a partition site and two proteins, ParA and ParB. To form the partition complex necessary for segregation, P1 ParB must recognize a complicated arrangement of A-box and B-box DNA motifs located on opposite ends of a sharply bent parS partition site of approximately 74 bp (refs 3-7). Here we describe structures of ParB bound to partition sites. ParB forms an asymmetric dimer with extended amino-terminal HTH (helix-turn-helix) domains that contact A-boxes. The two HTH domains emanate from a dimerized DNA-binding module composed of a six-stranded beta-sheet coiled-coil that binds B-boxes. Strikingly, these individual DNA-binding modules rotate freely about a flexible linker, enabling them to contact several arrangements of A- and B-boxes. Most notably, each DNA-binding element binds to and thus bridges adjacent DNA duplexes. These unique structural features of ParB explain how this protein can bind complex arrays of A- and B-box elements on adjacent DNA arms of the looped partition site.

Base Sequence↗
PubMed2006

Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB.

Nucleic acid damage by environmental and endogenous alkylation reagents creates lesions that are both mutagenic and cytotoxic, with the latter effect accounting for their widespread use in clinical cancer chemotherapy. Escherichia coli AlkB and the homologous human proteins ABH2 and ABH3 (refs 5, 7) promiscuously repair DNA and RNA bases damaged by S(N)2 alkylation reagents, which attach hydrocarbons to endocyclic ring nitrogen atoms (N1 of adenine and guanine and N3 of thymine and cytosine). Although the role of AlkB in DNA repair has long been established based on phenotypic studies, its exact biochemical activity was only elucidated recently after sequence profile analysis revealed it to be a member of the Fe-oxoglutarate-dependent dioxygenase superfamily. These enzymes use an Fe(II) cofactor and 2-oxoglutarate co-substrate to oxidize organic substrates. AlkB hydroxylates an alkylated nucleotide base to produce an unstable product that releases an aldehyde to regenerate the unmodified base. Here we have determined crystal structures of substrate and product complexes of E. coli AlkB at resolutions from 1.8 to 2.3 A. Whereas the Fe-2-oxoglutarate dioxygenase core matches that in other superfamily members, a unique subdomain holds a methylated trinucleotide substrate into the active site through contacts to the polynucleotide backbone. Amide hydrogen exchange studies and crystallographic analyses suggest that this substrate-binding 'lid' is conformationally flexible, which may enable docking of diverse alkylated nucleotide substrates in optimal catalytic geometry. Different crystal structures show open and closed states of a tunnel putatively gating O2 diffusion into the active site. Exposing crystals of the anaerobic Michaelis complex to air yields slow but substantial oxidation of 2-oxoglutarate that is inefficiently coupled to nucleotide oxidation. These observations suggest that protein dynamics modulate redox chemistry and that a hypothesized migration of the reactive oxy-ferryl ligand on the catalytic Fe ion may be impeded when the protein is constrained in the crystal lattice.

Alkylation↗
PubMed2006

Protein flexibility acclimatizes photosynthetic energy conversion to the ambient temperature.

Adjustment of catalytic activity in response to diverse ambient temperatures is fundamental to life on Earth. A crucial example of this is photosynthesis, where solar energy is converted into electrochemical potential that drives oxygen and biomass generation at temperatures ranging from those of frigid Antarctica to those of scalding hot springs. The energy conversion proceeds by concerted mobilization of electrons and protons on photoexcitation of reaction centre protein complexes. Following physicochemical paradigms, the rates of imperative steps in this process were predicted to increase exponentially with rising temperatures, resulting in different yields of solar energy conversion at the distinct growth temperatures of photosynthetic mesophiles and extremophiles. In contrast, here we show a meticulous adjustment of energy conversion rate, resulting in similar yields from mesophiles and thermophiles. The key molecular players in the temperature adjustment process consist of a cluster of hitherto unrecognized protein cavities and an adjacent packing motif that jointly impart local flexibility crucial to the reaction centre proteins. Mutations within the packing motif of mesophiles that increase the bulkiness of the amino-acid side chains, and thus reduce the size of the cavities, promote thermophilic behaviour. This novel biomechanical mechanism accounts for the slowing of the catalytic reaction above physiological temperatures in contradiction to the classical Arrhenius paradigm. The mechanism provides new guidelines for manipulating the acclimatization of enzymes to the ambient temperatures of diverse habitats. More generally, it reveals novel protein elements that are of potential significance for modulating structure-activity relationships in membrane and globular proteins alike.

Acclimatization↗
PubMed2006

Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery.

Protein ubiquitination is a common form of post-translational modification that regulates a broad spectrum of protein substrates in diverse cellular pathways. Through a three-enzyme (E1-E2-E3) cascade, the attachment of ubiquitin to proteins is catalysed by the E3 ubiquitin ligase, which is best represented by the superfamily of the cullin-RING complexes. Conserved from yeast to human, the DDB1-CUL4-ROC1 complex is a recently identified cullin-RING ubiquitin ligase, which regulates DNA repair, DNA replication and transcription, and can also be subverted by pathogenic viruses to benefit viral infection. Lacking a canonical SKP1-like cullin adaptor and a defined substrate recruitment module, how the DDB1-CUL4-ROC1 E3 apparatus is assembled for ubiquitinating various substrates remains unclear. Here we present crystallographic analyses of the virally hijacked form of the human DDB1-CUL4A-ROC1 machinery, which show that DDB1 uses one beta-propeller domain for cullin scaffold binding and a variably attached separate double-beta-propeller fold for substrate presentation. Through tandem-affinity purification of human DDB1 and CUL4A complexes followed by mass spectrometry analysis, we then identify a novel family of WD40-repeat proteins, which directly bind to the double-propeller fold of DDB1 and serve as the substrate-recruiting module of the E3. Together, our structural and proteomic results reveal the structural mechanisms and molecular logic underlying the assembly and versatility of a new family of cullin-RING E3 complexes.

Amino Acid Motifs↗
PubMed2006

The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA.

Influenza A viruses pose a serious threat to world public health, particularly the currently circulating avian H5N1 viruses. The influenza viral nucleoprotein forms the protein scaffold of the helical genomic ribonucleoprotein complexes, and has a critical role in viral RNA replication. Here we report a 3.2 A crystal structure of this nucleoprotein, the overall shape of which resembles a crescent with a head and a body domain, with a protein fold different compared with that of the rhabdovirus nucleoprotein. Oligomerization of the influenza virus nucleoprotein is mediated by a flexible tail loop that is inserted inside a neighbouring molecule. This flexibility in the tail loop enables the nucleoprotein to form loose polymers as well as rigid helices, both of which are important for nucleoprotein functions. Single residue mutations in the tail loop result in the complete loss of nucleoprotein oligomerization. An RNA-binding groove, which is found between the head and body domains at the exterior of the nucleoprotein oligomer, is lined with highly conserved basic residues widely distributed in the primary sequence. The nucleoprotein structure shows that only one of two proposed nuclear localization signals are accessible, and suggests that the body domain of nucleoprotein contains the binding site for the viral polymerase. Our results identify the tail loop binding pocket as a potential target for antiviral development.

Amino Acid Sequence↗
PubMed2006

Negative normal stress in semiflexible biopolymer gels.

When subject to stress or external loads, most materials resist deformation. Any stable material, for instance, resists compression-even liquids. Solids also resist simple shear deformations that conserve volume. Under shear, however, most materials also have a tendency to expand in the direction perpendicular to the applied shear stress, a response that is known as positive normal stress. For example, wet sand tends to dilate when sheared, and therefore dries around our feet when we walk on the beach. In the case of simple solids, elastic rods or wires tend to elongate when subject to torsion. Here, we show that networks of semiflexible biopolymers such as those that make up both the cytoskeleton of cells and the extracellular matrix exhibit the opposite tendency: when sheared between two plates, they tend to pull the plates together. We show that these negative normal stresses can be as large as the shear stress and that this property is directly related to the nonlinear strain-stiffening behaviour of biopolymer gels.

Biopolymers↗
PubMed2001

The role of conformational flexibility in prion propagation and maintenance for Sup35p.

The [PSI(+)] factor of Saccharomyces cerevisiae is a protein-based genetic element (prion) comprised of a heritable altered conformation of the cytosolic translation termination factor Sup35p. In vitro, the prion-determining region (NM) of Sup35p undergoes conformational conversion from a highly flexible soluble state to structured amyloid fibers, with a rate that is greatly accelerated by preformed NM fiber nuclei. Nucleated conformational conversion is the molecular basis of the genetic inheritance of [PSI(+)] and provides a new model for studying amyloidogenesis. Here we investigate the importance of structure and structural flexibility in soluble NM. Elevated temperatures, chemical chaperones and certain mutations in NM increase or change its structural content and inhibit or enhance nucleated conformational conversion. We propose that the structural flexibility of NM is particularly suited to allowing heritable protein-based changes in cellular behavior.

Amyloidosis↗
PubMed2001

Structural insight into Parkinson's disease treatment from drug-inhibited DOPA decarboxylase.

DOPA decarboxylase (DDC) is responsible for the synthesis of the key neurotransmitters dopamine and serotonin via decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan, respectively. DDC has been implicated in a number of clinic disorders, including Parkinson's disease and hypertension. Peripheral inhibitors of DDC are currently used to treat these diseases. We present the crystal structures of ligand-free DDC and its complex with the anti-Parkinson drug carbiDOPA. The inhibitor is bound to the enzyme by forming a hydrazone linkage with the cofactor, and its catechol ring is deeply buried in the active site cleft. The structures provide the molecular basis for the development of new inhibitors of DDC with better pharmacological characteristics.

5-Hydroxytryptophan↗
PubMed2001

Solution structure of Ca(2+)-calmodulin reveals flexible hand-like properties of its domains.

The solution structure of Ca(2+)-ligated calmodulin is determined from residual dipolar couplings measured in a liquid crystalline medium and from a large number of heteronuclear J couplings for defining side chains. Although the C-terminal domain solution structure is similar to the X-ray crystal structure, the EF hands of the N-terminal domain are considerably less open. The substantial differences in interhelical angles correspond to negligible changes in short interproton distances and, therefore, cannot be identified by comparison of NOEs and X-ray data. NOE analysis, however, excludes a two-state equilibrium in which the closed apo conformation is partially populated in the Ca(2+)-ligated state. The difference between the crystal and solution structures of Ca(2+)-calmodulin indicates considerable backbone plasticity within the domains of calmodulin, which is key to their ability to bind a wide range of targets. In contrast, the vast majority of side chains making up the target binding surface are locked into the same chi(1) rotameric states as in complexes with target peptide.

Animals↗
PubMed2002

A new FAD-binding fold and intersubunit disulfide shuttle in the thiol oxidase Erv2p.

Erv2p is an FAD-dependent sulfhydryl oxidase that can promote disulfide bond formation during protein biosynthesis in the yeast endoplasmic reticulum. The structure of Erv2p, determined by X-ray crystallography to 1.5 A resolution, reveals a helix-rich dimer with no global resemblance to other known FAD-binding proteins or thiol oxidoreductases. Two pairs of cysteine residues are required for Erv2p activity. The first (Cys-Gly-Glu-Cys) is adjacent to the isoalloxazine ring of the FAD. The second (Cys-Gly-Cys) is part of a flexible C-terminal segment that can swing into the vicinity of the first cysteine pair in the opposite subunit of the dimer and may shuttle electrons between substrate protein dithiols and the FAD-proximal disulfide.

Amino Acid Motifs↗
PubMed2002

Structural insights into CPT-11 activation by mammalian carboxylesterases.

Mammalian carboxylesterases cleave the anticancer prodrug CPT-11 (Irinotecan) into SN-38, a potent topoisomerase I poison, and 4-piperidino-piperidine (4PP). We present the 2.5 A crystal structure of rabbit liver carboxylesterase (rCE), the most efficient enzyme known to activate CPT-11 in this manner, in complex with the leaving group 4PP. 4PP is observed bound adjacent to a high-mannose Asn-linked glycosylation site on the surface of rCE. This product-binding site is separated from the catalytic gorge by a thin wall of amino acid side chains, suggesting that 4PP may be released through this secondary product exit pore. The crystallographic observation of a leaving group bound on the surface of rCE supports the 'back door' product exit site proposed for the acetylcholinesterases. These results may facilitate the design of improved anticancer drugs or enzymes for use in viral-directed cancer cotherapies.

Amino Acid Sequence↗
PubMed2006

Close membrane-membrane proximity induced by Ca(2+)-dependent multivalent binding of synaptotagmin-1 to phospholipids.

Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.

Animals↗