Conformations of immunoglobulin hypervariable regions

On the basis of comparative studies of known antibody structures and sequences it has been argued that there is a small repertoire of main-chain conformations for at least five of the six hypervariable regions of antibodies, and that the particular conformation adopted is determined by a few key conserved residues. These hypotheses are now supported by reasonably successful predictions of the structures of most hypervariable regions of various antibodies, as revealed by comparison with their subsequently determined structures.     

Antigenized antibodies.

A new process, antigenization of antibodies, consisting of the expression of oligopeptides in the hypervariable loops of an antibody molecule is described. The potential applications of antigenized antibodies are discussed.     

The transorientation hypothesis for codon recognition during protein synthesis

During decoding, a codon of messenger RNA is matched with its cognate aminoacyl-transfer RNA and the amino acid carried by the tRNA is added to the growing protein chain. Here we propose a molecular mechanism for the decoding phase of translation: the transorientation hypothesis. The model incorporates a newly identified tRNA binding site and utilizes a flip between two tRNA anticodon loop structures, the 5'-stacked and the 3'-stacked conformations. The anticodon loop acts as a three-dimensional hinge permitting rotation of the tRNA about a relatively fixed codon-anticodon pair. This rotation, driven by a conformational change in elongation factor Tu involving GTP hydrolysis, transorients the incoming tRNA into the A site from the D site of initial binding and decoding, where it can be proofread and accommodated. The proposed mechanisms are compatible with the known structures, conformations and functions of the ribosome and its component parts including tRNAs and EF-Tu, in both the GTP and GDP states.     

Replacing the complementarity-determining regions in a human antibody with those from a mouse

The variable domains of an antibody consist of a beta-sheet framework with hypervariable regions (or complementarity-determining regions--CDRs) which fashion the antigen-binding site. Here we attempted to determine whether the antigen-binding site could be transplanted from one framework to another by grafting the CDRs. We substituted the CDRs from the heavy-chain variable region of mouse antibody B1-8, which binds the hapten NP-cap (4-hydroxy-3-nitrophenacetyl caproic acid; KNP-cap = 1.2 microM), for the corresponding CDRs of a human myeloma protein. We report that in combination with the B1-8 mouse light chain, the new antibody has acquired the hapten affinity of the B1-8 antibody (KNP-cap = 1.9 microM). Such 'CDR replacement' may offer a means of constructing human monoclonal antibodies from the corresponding mouse monoclonal antibodies.     

Three-dimensional structure of an idiotope-anti-idiotope complex.

Serologically detected antigenic determinants unique to an antibody or group of antibodies are called idiotopes. The sum of idiotopes of an antibody constitute its idiotype. Idiotypes have been intensively studied following a hypothesis for the self-regulation of the immune system through a network of idiotype-anti-idiotype interactions. Furthermore, as antigen and anti-idiotypes can competitively bind to idiotype-positive, antigen-specific antibodies, anti-idiotypes may carry an 'internal image' of the external antigen. Here we describe the structure of the complex between the monoclonal anti-lysozyme FabD1.3 and the anti-idiotopic FabE225 at 2.5 A resolution. This complex defines a private idiotope consisting of 13 amino-acid residues, mainly from the complementarity-determining regions of D1.3. Seven of these residues make contacts with the antigen, indicating a significant overlap between idiotope and antigen-combining site. Idiotopic mimicry of the external antigen is not achieved at the molecular level in this example.     

Rational screenning in combinatorial peptide libraries of protein functional loop

Redesigning the sequences of protein loops is a frequent practice in protein design. Based on the new results of protein loop database analysis, a rational computer simulation strategy is proposed to obtain functional proteins, which exploits a fast and accurate program to calculate the protein loop conformation, and at the same time, combines molecular docking method with combinatorial chemistry strategy to screen the combinatorial peptide library of protein loops. A characteristic of this method is that it separates the conformation computation of backbone from that of side chain and incorporates side chain growth into the docking procedure and therefore greatly reduces the computation by converting the huge computation on explosive conformations to relatively small computation on limited canonical backbone structures and side chain growth. This method can be practically used in screening combinatorial peptide libraries of protein loops.     

多分散高分子固液界面吸附构型的Monte Carlo模拟

在格子模型基础上用M onte Carlo方法模拟研究了多分散高分子在固液界面的吸附行为,重点考察了平均分布和正态分布两种不同链长分布形式的高分子在固液界面吸附构型的分布规律。发现高分子不同的链长分布形式,对高分子吸附构型的性质影响较大。正态分布的高分子体系中高分子的3种吸附构型(T ails,Loops和T rains)的浓度和数目比相同条件下平均分布的高分子体系内要低得多。特别是当高分子链节吸附能较低时,两者的差别非常大。平均分布的高分子体系高分子吸附构型对温度和高分子总链节浓度的变化更加敏感。T ails构型由于受到高分子链节热运动以及吸附层压缩作用的影响,在高温或高吸附作用能下,其密度分布表现出和其他两种吸附构型完全不同的形式。温度、高分子链节吸附作用能以及高分子总链节浓度对3种吸附构型的影响和单分散体系趋势一致,但是存在着定量的差别。     

Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9

Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded β-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which-with PG9-involves a site of vulnerability comprising just two glycans and a strand.     

Reshaping human antibodies for therapy

A human IgGI antibody has been reshaped for serotherapy in humans by introducing the six hypervariable regions from the heavy- and light-chain variable domains of a rat antibody directed against human lymphocytes. The reshaped human antibody is as effective as the rat antibody in complement and is more effective in cell-mediated lysis of human lymphocytes.     

Crystal structures of the membrane-binding C2 domain of human coagulation factor V

Rapid and controlled clot formation is achieved through sequential activation of circulating serine proteinase precursors on phosphatidylserine-rich procoagulant membranes of activated platelets and endothelial cells. The homologous complexes Xase and prothrombinase, each consisting of an active proteinase and a non-enzymatic cofactor, perform critical steps within this coagulation cascade. The activated cofactors VIIIa and Va, highly specific for their cognate proteinases, are each derived from precursors with the same A1-A2-B-A3-C1-C2 architecture. Membrane binding is mediated by the C2 domains of both cofactors. Here we report two crystal structures of the C2 domain of human factor Va. The conserved beta-barrel framework provides a scaffold for three protruding loops, one of which adopts markedly different conformations in the two crystal forms. We propose a mechanism of calcium-independent, stereospecific binding of factors Va and VIIIa to phospholipid membranes, on the basis of (1) immersion of hydrophobic residues at the apices of these loops in the apolar membrane core; (2) specific interactions with phosphatidylserine head groups in the groove enclosed by these loops; and (3) favourable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups.     

Structure of an unliganded simian immunodeficiency virus gp120 core

Envelope glycoproteins of human and simian immunodeficiency virus (HIV and SIV) undergo a series of conformational changes when they interact with receptor (CD4) and co-receptor on the surface of a potential host cell, leading ultimately to fusion of viral and cellular membranes. Structures of fragments of gp120 and gp41 from the envelope protein are known, in conformations corresponding to their post-attachment and postfusion states, respectively. We report the crystal structure, at 4 A resolution, of a fully glycosylated SIV gp120 core, in a conformation representing its prefusion state, before interaction with CD4. Parts of the protein have a markedly different organization than they do in the CD4-bound state. Comparison of the unliganded and CD4-bound structures leads to a model for events that accompany receptor engagement of an envelope glycoprotein trimer. The two conformations of gp120 also present distinct antigenic surfaces. We identify the binding site for a compound that inhibits viral entry.     

3D Simulation of Two-bar Warp-knitted Structures

In this study,3D computer modeling of simple warp-knitted structures is achieved based on 3D model of warp-knitted loops.Firstly, according to the studying on the geometric structure of warp-knitted loops, Gktepe's 3D solid yarn model is developed,and the dimensions of the warp-knitted loops are obtained;then 3D models of stitch defined by eleven given points and in-lay defined by five given points are built with the method of Non-Uniform Rational B-Spline (NURBS) curves and surfaces.Secondly, according to...     

丙氨酸三肽的稳定构象的探究

探究快速地获得丙氨酸三肽的稳定构象。利用Gaussian09程序包中的 H F/6-31G （d ，p ）方法得到丙氨酸三肽势能面。固定二面角φ2、ψ2的度数，改变二面角φ1、ψ1，计算了丙氨酸三肽不同结构的相对能量，这些结构的相对能量的图像趋势大致相同，可知两组二面角之间影响不大。使用丙氨酸二肽稳定构象所对应的骨架二面角的组合，获得丙氨酸三肽的初始构型，使用B3LYP/6-31G（d ，p）方法优化获得其稳定构象。这种方法可为多肽分子寻找稳定构象提供1种简单而快速的方法。     

Quadruplex structure of Oxytricha telomeric DNA oligonucleotides.

The telomeres of most eukaryotes contain a repeating G-rich sequence with the consensus d(T/A)1-4G1-8, of which 12-16 bases form a 3' single-strand overhang beyond the telomeric duplex. It has been proposed that these G-rich oligonucleotides associate to form four-stranded structures from one, two or four individual strands and that these structures may be relevant in vivo. The proposed structures contain Hoogsteen base-paired G-quartets, precedent for which has been in the literature for many years. Here we use 1H NMR spectroscopy to study the conformations of the DNA oligonucleotides d(G4T4G4) (Oxy-1.5) and d(G4T4G4T4G4T4G4) (Oxy-3.5) which contain the Oxytricha telomere repeat (T4G4). We find that these molecules fold to form a symmetrical bimolecular and an intramolecular quadruplex, respectively. Both structures have four G-quartets formed from nucleotides that are alternately syn and anti along each strand. This arrangement differs from earlier models in which the strands are alternately all syn or all anti. The T4 loops in Oxy-1.5 are on opposite ends of the quadruplex and loop diagonally across the G-quartet, resulting in adjacent strands being alternately parallel and antiparallel.     

Beta-hairpin families in globular proteins

Beta-hairpins, one of the simplest supersecondary structures, are widespread in globular proteins, and have often been suggested as possible sites for nucleation. Here we consider the conformation and sequences of the loop regions of beta-hairpins by analysing proteins of known structure. We find that the 'tight' beta-hairpins, classified by the length and conformations of their loop regions, form distinct families and that the loop regions of the family members have sequences which are characteristic of that family. The two-residue hairpin loops include almost entirely I' or II' beta-turns, in contrast to the general preference for type I and type II turns. These findings are being used to help define templates or consensus sequences to be incorporated into our existing supersecondary structure prediction algorithm. This information can also be used in model-building homologous proteins.     

Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli

In antibodies, a heavy and a light chain variable domain, VH and VL, respectively, pack together and the hypervariable loops on each domain contribute to binding antigen. We find, however, that isolated VH domains with good antigen-binding affinities can also be prepared. Using the polymerase chain reaction, diverse libraries of VH genes were cloned from the spleen genomic DNA of mice immunized with either lysozyme or keyhole-limpet haemocyanin. From these libraries, VH domains were expressed and secreted from Escherichia coli. Binding activities were detected against both antigens, and two VH domains were characterized with affinities for lysozyme in the 20 nM range. Isolated variable domains may offer an alternative to monoclonal antibodies and serve as the key to building high-affinity human antibodies. We suggest the name 'single domain antibodies (dAbs)' for these antigen binding demands.     

Molecular motions and conformational transition between different conformational states of HIV-1 gp 120 envelope glycoprotein

The HIV-1 gp120 exterior envelope glycoprotein undergoes a series of conformational rearrangements while sequentially interacting with the receptor CD4 and coreceptor CCR5 or CXCR4 on the surface of host cells to initiate virus entry. Both the crystal structures of the HIV-1 gp120 core bound by the CD4 and antigen 17b and the SIV gp120 core pre-bound by CD4 are known. Despite the wealth of knowledge on these static snapshots of molecular conformations,the details of molecular motions involved in conformational transition that are crucial to intervention remain elusive. We presented comprehensive comparative analyses of the dynamics behaviors of the gp120 in its CD4-complexed,CD4-free and CD4-unliganded states based on the homology models with modeled V3 and V4 loops by means of CONCOORD computer simulation to generate ensembles of feasible protein structures that were sub-sequently analysed by essential dynamics analyses to identify preferred concerted motions. The re-vealed collective fluctuations are dominated by complex modes of combinational motions of the rota-tion/twisting,flexing/closure,and shortness/elongation between or within the inner,outer,and bridg-ing-sheet domains,and these modes are related to the CD4 association and HIV neutralization avoid-ance. Further essential subspace overlap analyses were performed to quantitatively distinguish the preference for conformational transitions between the three states,revealing that the unliganded gp120 has a greater potential to translate its conformation into the conformational state adopted by the CD4-complexed gp120 than by the CD4-free gp120,whereas the CD4-free gp120 has a greater potential to translate its conformation into the unliganded state than the CD4-complexed gp120 does. These dynamics data of gp120 in its different conformations are helpful in understanding the relationship between the molecular motion/conformational transition and the function of gp120,and in gp120-structure-based subunit vaccine design.     

基于裂纹尖端张开角准则的多裂纹薄壁结构剩余强度分析

评估由中央裂纹（M(T)）试样获得的裂纹尖端张开角(CTOA)预测多裂纹薄壁结构剩余强度的有效性.利用ABAQUS软件建立含有M(T)试样的二维有限元模型,采用裂纹尖端张开角断裂准则和平面应力模型模拟其裂纹扩展过程.对具有不同初始裂纹长度的M(T)试样,该方法预测的剩余强度和载荷-裂纹扩展增量曲线与试验结果接近,误差小于6%;但对于含有多条裂纹试样的剩余强度预测值偏大,相对误差在20%左右.通过增加对裂纹尖端周围单元约束的改进,采用平面应变核模型可以有效提高剩余强度预测精度;对具有不同多裂纹构型的薄壁结构进行分析,采用恒定裂纹尖端张开角和平面应变核的方法均可以得到与试验相近的结果,剩余强度预测误差基本都在10%以内.