Beta-helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect

Insect antifreeze proteins (AFP) are considerably more active at inhibiting ice crystal growth than AFP from fish or plants. Several insect AFPs, also known as thermal hysteresis proteins, have been cloned and expressed. Their maximum activity is 3-4 times that of fish AFPs and they are 10-100 times more effective at micromolar concentrations. Here we report the solution structure of spruce budworm (Choristoneura fumiferana) AFP and characterize its ice-binding properties. The 9-kDa AFP is a beta-helix with a triangular cross-section and rectangular sides that form stacked parallel beta-sheets; a fold which is distinct from the three known fish AFP structures. The ice-binding side contains 9 of the 14 surface-accessible threonines organized in a regular array of TXT motifs that match the ice lattice on both prism and basal planes. In support of this model, ice crystal morphology and ice-etching experiments are consistent with AFP binding to both of these planes and thus may explain the greater activity of the spruce budworm antifreeze.     

猪皮明胶抗冻多肽的制备及其低温保护活性研究

以食品源猪皮明胶为原料,用碱性蛋白酶水解,制备抗冻多肽,并对影响碱性蛋白酶水解的各个因素进行研究. 以水解制备的多肽对过氧化氢酶的低温保护活性为指标,在单因素实验的基础上,通过正交实验确定碱性蛋白酶水解猪皮明胶的最适pH为9.0,最适温度是37℃,酶和底物比例为1∶50,酶解时间3h. 在此条件下制备的抗冻多肽对过氧化氢酶的低温保护活性为88.2%. 与牛血清蛋白和商业抗冻剂（4%蔗糖+4%山梨醇）比较,抗冻多肽表现出更优越的抗冻活性,且其抗冻活性和浓度成正相关关系.     

Crystal structure of opsin in its G-protein-interacting conformation

Opsin, the ligand-free form of the G-protein-coupled receptor rhodopsin, at low pH adopts a conformationally distinct, active G-protein-binding state known as Ops*. A synthetic peptide derived from the main binding site of the heterotrimeric G protein-the carboxy terminus of the alpha-subunit (GalphaCT)-stabilizes Ops*. Here we present the 3.2 A crystal structure of the bovine Ops*-GalphaCT peptide complex. GalphaCT binds to a site in opsin that is opened by an outward tilt of transmembrane helix (TM) 6, a pairing of TM5 and TM6, and a restructured TM7-helix 8 kink. Contacts along the inner surface of TM5 and TM6 induce an alpha-helical conformation in GalphaCT with a C-terminal reverse turn. Main-chain carbonyl groups in the reverse turn constitute the centre of a hydrogen-bonded network, which links the two receptor regions containing the conserved E(D)RY and NPxxY(x)(5,6)F motifs. On the basis of the Ops*-GalphaCT structure and known conformational changes in Galpha, we discuss signal transfer from the receptor to the G protein nucleotide-binding site.     

Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis

The hairpin ribozyme catalyses sequence-specific cleavage of RNA. The active site of this natural RNA results from the docking of two irregular helices: stems A and B. One strand of stem A harbours the scissile bond. The 2.4 A resolution structure of a hairpin ribozyme-inhibitor complex reveals that the ribozyme aligns the 2'-OH nucleophile and the 5'-oxo leaving group by twisting apart the nucleotides that flank the scissile phosphate. The base of the nucleotide preceding the cleavage site is stacked within stem A; the next nucleotide, a conserved guanine, is extruded from stem A and accommodated by a highly complementary pocket in the minor groove of stem B. Metal ions are absent from the active site. The bases of four conserved purines are positioned potentially to serve as acid-base catalysts. This is the first structure determination of a fully assembled ribozyme active site that catalyses a phosphodiester cleavage without recourse to metal ions.     

Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications

The APOBEC family members are involved in diverse biological functions. APOBEC3G restricts the replication of human immunodeficiency virus (HIV), hepatitis B virus and retroelements by cytidine deamination on single-stranded DNA or by RNA binding. Here we report the high-resolution crystal structure of the carboxy-terminal deaminase domain of APOBEC3G (APOBEC3G-CD2) purified from Escherichia coli. The APOBEC3G-CD2 structure has a five-stranded beta-sheet core that is common to all known deaminase structures and closely resembles the structure of another APOBEC protein, APOBEC2 (ref. 5). A comparison of APOBEC3G-CD2 with other deaminase structures shows a structural conservation of the active-site loops that are directly involved in substrate binding. In the X-ray structure, these APOBEC3G active-site loops form a continuous 'substrate groove' around the active centre. The orientation of this putative substrate groove differs markedly (by 90 degrees) from the groove predicted by the NMR structure. We have introduced mutations around the groove, and have identified residues involved in substrate specificity, single-stranded DNA binding and deaminase activity. These results provide a basis for understanding the underlying mechanisms of substrate specificity for the APOBEC family.     

Crystal structure of an Eph receptor-ephrin complex.

The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are important in regulating cell-cell interactions as they initiate a unique bidirectional signal transduction cascade whereby information is communicated into both the Eph-expressing and the ephrin-expressing cells. Initially identified as regulators of axon pathfinding and neuronal cell migration, Ephs and ephrins are now known to have roles in many other cell-cell interactions, including those of vascular endothelial cells and specialized epithelia. Here we report the crystal structure of the complex formed between EphB2 and ephrin-B2, determined at 2.7 A resolution. Each Eph receptor binds an ephrin ligand through an expansive dimerization interface dominated by the insertion of an extended ephrin loop into a channel at the surface of the receptor. Two Eph-Ephrin dimers then join to form a tetramer, in which each ligand interacts with two receptors and each receptor interacts with two ligands. The Eph and ephrin molecules are precisely positioned and orientated in these complexes, promoting higher-order clustering and the initiation of bidirectional signalling.     

Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand

Target cell lysis is regulated by natural killer (NK) cell receptors that recognize class I MHC molecules. Here we report the crystal structure of the human immunoglobulin-like NK cell receptor KIR2DL2 in complex with its class I ligand HLA-Cw3 and peptide. KIR binds in a nearly orthogonal orientation across the alpha1 and alpha2 helices of Cw3 and directly contacts positions 7 and 8 of the peptide. No significant conformational changes in KIR occur on complex formation. The receptor footprint on HLA overlaps with but is distinct from that of the T-cell receptor. Charge complementarity dominates the KIR/HLA interface and mutations that disrupt interface salt bridges substantially diminish binding. Most contacts in the complex are between KIR and conserved HLA-C residues, but a hydrogen bond between Lys 44 of KIR2DL2 and Asn 80 of Cw3 confers the allotype specificity. KIR contact requires position 8 of the peptide to be a residue smaller than valine. A second KIR/HLA interface produced an ordered receptor-ligand aggregation in the crystal which may resemble receptor clustering during immune synapse formation.     

The structure of an oligo(dA).oligo(dT) tract and its biological implications

Poly(dA).poly(dT) has unusual properties in that it cannot associate into nucleosomes and short, phased runs of it cause DNA bending. The crystal structure of a B-type DNA dodecamer containing a homopolymeric run of six A.T base pairs shows that this region possesses special structural features, including a system of bifurcated hydrogen bonds, which explains some of the properties of this simple homopolymer.     

Crystal structure refinement of suolunite and its significance to the cement techniques

Based on the crystal structure refinement, the arrangement and characteristics of the double tetrahedra of Si-O backbone of suolunite have been precisely clarified. The double tetrahedra are isolated, and the orientations of the closely adjacent double tetrahedra are perpendicular to each other, which results in the poor cleavage and high hardness of suolunite. Compared with the crystal structure of synthesized C-S-H minerals in Portland cement hydrates, this mineral apparently avails the increasing of the compressive strength of cement materials and the manufaturing of high performance concrete. Thus this research provides a new idea for the optimization of Portland cement manufaturing techniques.     

Crystal structure of an H/ACA box ribonucleoprotein particle

H/ACA ribonucleoprotein particles (RNPs) are a family of RNA pseudouridine synthases that specify modification sites through guide RNAs. They also participate in eukaryotic ribosomal RNA processing and are a component of vertebrate telomerases. Here we report the crystal structure, at 2.3 A resolution, of an entire archaeal H/ACA RNP consisting of proteins Cbf5, Nop10, Gar1 and L7ae, and a single-hairpin H/ACA RNA, revealing a modular organization of the complex. The RNA upper stem is bound to a composite surface formed by L7ae, Nop10 and Cbf5, and the RNA lower stem and ACA signature motif are bound to the PUA domain of Cbf5, thereby positioning middle guide sequences so that they are primed to pair with substrate RNA. Furthermore, Gar1 may regulate substrate loading and release. The structure rationalizes the consensus structure of H/ACA RNAs, suggests a functional role of each protein, and provides a framework for understanding the mechanism of RNA-guided pseudouridylation, as well as various cellular functions of H/ACA RNP.     

Crystal structure of chaperone protein PapD reveals an immunoglobulin fold

The chaperone protein PapD mediates assembly of pili in Escherichia coli. Its polypeptide chain folds into two immunoglobulin-type domains that are homologous in sequence to the human lymphocyte differentiation antigen Leu-1/CD5.     

牦牛骨胶原蛋白肽的制备及其抗冻性能研究

以新鲜牦牛骨为原料,制备了牦牛骨胶原抗冻肽,并对其抗冻性能进行了研究。结果表明,以EDTA·2Na为脱钙剂对牛骨进行脱钙的最佳脱钙浓度为0.25 mol/L,脱钙时长为48 h,得到的胶原蛋白最大提取率为43.99%。胃蛋白酶酶解制备酶解复合物的最佳时间为48 h,温度25 ℃,得到的胶原蛋白的提取率可达48.53%。同时,采用改良的冰亲和吸附法对胶原抗冻肽进行吸附,得到最佳吸附时间为4 h,吸附温度为-2 ℃,吸附浓度为2 mg/ml,在此条件下,蛋白得率为48.1%,而热滞活性值可高达5.76 ℃。牦牛骨胶原抗冻肽属于一种高热滞活性蛋白资源,在食品冷冻保藏以及运输等领域有较好的应用前景。     

Crystal structure of a complex between anthrax toxin and its host cell receptor

Anthrax toxin consists of the proteins protective antigen (PA), lethal factor (LF) and oedema factor (EF). The first step of toxin entry into host cells is the recognition by PA of a receptor on the surface of the target cell. Subsequent cleavage of receptor-bound PA enables EF and LF to bind and form a heptameric PA63 pre-pore, which triggers endocytosis. Upon acidification of the endosome, PA63 forms a pore that inserts into the membrane and translocates EF and LF into the cytosol. Two closely related host cell receptors, TEM8 and CMG2, have been identified. Both bind to PA with high affinity and are capable of mediating toxicity. Here, we report the crystal structure of the PA-CMG2 complex at 2.5 A resolution. The structure reveals an extensive receptor-pathogen interaction surface mimicking the non-pathogenic recognition of the extracellular matrix by integrins. The binding surface is closely conserved in the two receptors and across species, but is quite different in the integrin domains, explaining the specificity of the interaction. CMG2 engages two domains of PA, and modelling of the receptor-bound PA63 heptamer suggests that the receptor acts as a pH-sensitive brace to ensure accurate and timely membrane insertion. The structure provides new leads for the discovery of anthrax anti-toxins, and should aid the design of cancer therapeutics.     

Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel

Voltage-gated sodium (Na(v)) channels are essential for the rapid depolarization of nerve and muscle, and are important drug targets. Determination of the structures of Na(v) channels will shed light on ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Na(v) channels, exemplified by the Na(+)-selective channel of bacteria (NaChBac), provides a useful model system for structure-function analysis. Here we report the crystal structure of Na(v)Rh, a NaChBac orthologue from the marine alphaproteobacterium HIMB114 (Rickettsiales sp. HIMB114; denoted Rh), at 3.05?? resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr?178 and Leu?179 constitute an inner site within the selectivity filter where a hydrated Ca(2+) resides in the crystal structure. The outer mouth of the Na(+) selectivity filter, defined by Ser?181 and Glu?183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation in which all the gating charges are exposed to the extracellular environment. We propose that Na(v)Rh is in an 'inactivated' conformation. Comparison of Na(v)Rh with Na(v)Ab reveals considerable conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.     

Crystal structure of an HIV-binding recombinant fragment of human CD4

CD4 glycoprotein on the surface of T cells helps in the immune response and is the receptor for HIV infection. The structure of a soluble fragment of CD4 determined at 2.3 A resolution reveals that the molecule has two intimately associated immunoglobulin-like domains. Residues implicated in HIV recognition by analysis of mutants and antibody binding are salient features in domain D1. Domain D2 is distinguished by a variation on the beta-strand topologies of antibody domains and by an intra-sheet disulphide bridge.     

Isolation, purification and characterization of secondary structure of antifreeze protein fromAmmopiptanthus mongolicus

Antifreeze protein (AFP) from Ammopiptanthus mongolicus leaves was isolated and purified with DEAE cellulose 52, Sephacryl 300, heat treatment and preparative isotachophoresis. The molecular weight of AFP was 50 ku by SDS-PAGE measurement. The average thermal hysteresis activity (THA) of AFP was 0.35℃ at 5 mg/mL using a differential scanning calorimeter (DSC). This AFP showed heated stability. From circular dichroism (CD) of AFP spectral data from 195—240 nm, a well-defined secondary structure of 11% a-helix, 34% antiparallel b-sheet and 55% random coil for the plant AFP was deduced.