The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport

Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). The approximately 3.0 angstrom structure contains 14 transmembrane (TM) helices in an inward-facing conformation with a core structure of inverted repeats of 5 TM helices (TM2 to TM6 and TM7 to TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to that of the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.     

Crystal structure of Escherichia coli MscS,a voltage-modulated and mechanosensitive channel

The mechanosensitive channel of small conductance (MscS) responds both to stretching of the cell membrane and to membrane depolarization. The crystal structure at 3.9 angstroms resolution demonstrates that Escherichia coli MscS folds as a membrane-spanning heptamer with a large cytoplasmic region. Each subunit contains three transmembrane helices (TM1, -2, and -3), with the TM3 helices lining the pore, while TM1 and TM2, with membrane-embedded arginines, are likely candidates for the tension and voltage sensors. The transmembrane pore, apparently captured in an open state, connects to a large chamber, formed within the cytoplasmic region, that connects to the cytoplasm through openings that may function as molecular filters. Although MscS is likely to be structurally distinct from other ion channels, similarities in gating mechanisms suggest common structural elements.     

Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution

The crystal structure of the complex between Eco RI endonuclease and the cognate oligonucleotide TCGCGAATTCGCG provides a detailed example of the structural basis of sequence-specific DNA-protein interactions. The structure was determined, to 3 A resolution, by the ISIR (iterative single isomorphous replacement) method with a platinum isomorphous derivative. The complex has twofold symmetry. Each subunit of the endonuclease is organized into an alpha/beta domain consisting a five-stranded beta sheet, alpha helices, and an extension, called the "arm," which wraps around the DNA. The large beta sheet consists of antiparallel and parallel motifs that form the foundations for the loops and alpha helices responsible for DNA strand scission and sequence-specific recognition, respectively. The DNA cleavage site is located in a cleft that binds the DNA backbone in the vicinity of the scissile bond. Sequence specificity is mediated by 12 hydrogen bonds originating from alpha helical recognition modules. Arg200 forms two hydrogen bonds with guanine while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. These interactions discriminate the Eco RI hexanucleotide GAATTC from all other hexanucleotides because any base substitution would require rupture of at least one of these hydrogen bonds.     

Side chain contributions to the stability of alpha-helical structure in peptides

Short peptides that contain significant alpha-helical structure in aqueous solution allow the investigation of the role of amino acid side chains in stabilizing or destabilizing alpha-helix structure. A host-guest system of soluble synthetic peptides was designed that consisted of chains with the block sequence TyrSerGlu4Lys4X3Glu4Lys4, denoted EXK, in which X represents any "guest" amino acid residue. Circular dichroism spectroscopy indicates that the extent of helicity of these peptides follows the order Ala greater than Leu greater than Met greater than Gln greater than Ile greater than Val greater than Ser greater than Thr greater than Asn greater than Gly. This order differs from both host-guest copolymer values (Met greater than Ile greater than Leu greater than Ala greater than Gln greater than Val greater than Thr greater than Asn greater than Ser greater than Gly) and the tendencies of these amino acids to occur in helices in globular proteins (Ala greater than Met greater than Leu greater than Gln greater than Ile greater than Val greater than Asn, Thr greater than Ser greater than Gly), but matches the order found in a series of synthetic coiled-coil alpha helices, except for Ser. Proton nuclear magnetic resonance analysis of several EXK peptides indicates that these peptides are partially helical, with the helical residues favoring the amino terminus.     

猪Ⅱ型圆环病毒江苏株全基因进化及ORF3蛋白的生物信息学分析

对NCBI已经登记的国内分离的PCV2江苏株(GQ358994.1)进行序列比对及进化分析,并对其ORF3编码蛋白的氨基酸序列进行生物信息学方法分析,对其组分、理化性质、跨膜结构域、二级结构、糖基化位点、磷酸化位点和B细胞抗原表位进行预测和推断。结果表明,PCV2江苏株与国内分离株序列同源性极高(>99%),ORF3蛋白无跨膜区域,二级结构中自由卷曲含量最高,为54.81%,不含有糖基化位点,但有8个磷酸化位点。综合分析得出,ORF3蛋白具有大量的抗原决定簇。通过对ORF3蛋白进行生物信息学方法分析,为PRRSV疫苗设计提供理论基础。     

抗鸡传染性法氏囊病病毒单克隆抗体识别的抗原表位分析

用鸡传染性法氏囊病病毒(IBDV)及其重组表达蛋白VP2,VP3和单抗做各种ELISA试验,测定单抗识别的IBDV蛋白抗原及其多肽片段,并分析单抗识别IBDV抗原表位的空间关系。结果表明,抗IBDV单抗A,B,C,D,E,G与重组表达蛋白VP2呈阳性反应,而与表达蛋白VP3呈阴性反应,单抗C与表达蛋白VP2和VP3均呈阳性反应;单抗A,B,D,E,G识别IBDV VP2蛋白上相近或相似的抗原表位,均识别VP2蛋白多肽序列PJ1,PJ2,PJ5,PJ14,属于同一类群的构像性单克隆抗体。     

Limit of T cell tolerance to self proteins by peptide presentation

Cytotoxic T lymphocytes (CTLs) recognize foreign peptides bound to major histocompatibility complex (MHC) class I molecules. MHC molecules can also bind endogenous self peptides, to which T cells are tolerant. Normal mice contained CTLs specific for self peptides that were from proteins of ubiquitous or tissue-restricted expression. In vivo, these endogenous self peptides are not naturally presented in sufficient density by somatic cells expressing MHC class I molecules. They can, however, be presented if added exogenously. Thus, our data imply that CTLs are only tolerant of those endogenous self peptide sequences that are presented by MHC class I-positive cells in a physiological manner.     

Partial symmetrization of the photosynthetic reaction center

The bacterial photosynthetic reaction center (RC) is a pigmented intrinsic membrane protein that performs the primary charge separation event of photosynthesis, thereby converting light to chemical energy. The RC pigments are bound primarily by two homologous peptides, the L and M subunits, each containing five transmembrane helices. These alpha helices and pigments are arranged in an approximate C2 symmetry and form two possible electron transfer pathways. Only one of these pathways is actually used. In an attempt to identify nonhomologous residues that are responsible for functional differences between the two branches, homologous helical regions that interact extensively with the pigments were genetically symmetrized (that is, exchanged). For example, replacement of the fourth transmembrane helix (D helix) in the M subunit with the homologous helix from the L subunit yields photosynthetically inactive RCs lacking a critical photoactive pigment. Photosynthetic revertants have been isolated in which single amino acid substitutions (intragenic suppressors) compensate for this partial symmetrization.     

A structural mechanism for MscS gating in lipid bilayers

The mechanosensitive channel of small conductance (MscS) is a key determinant in the prokaryotic response to osmotic challenges. We determined the structural rearrangements associated with MscS activation in membranes, using functorial measurements, electron paramagnetic resonance spectroscopy, and computational analyses. MscS was trapped in its open conformation after the transbilayer pressure profile was modified through the asymmetric incorporation of lysophospholipids. The transition from the closed to the open state is accompanied by the downward tilting of the transmembrane TM1-TM2 hairpin and by the expansion, tilt, and rotation of the TM3 helices. These movements expand the permeation pathway, leading to an increase in accessibility to water around TM3. Our open MscS model is compatible with single-channel conductance measurements and supports the notion that helix tilting is associated with efficient pore widening in mechanosensitive channels.     

Defective presentation of endogenous antigen by a cell line expressing class I molecules

Cytotoxic T lymphocytes (CTLs) recognize class I major histocompatibility complex (MHC) molecules associated with antigenic peptides derived from endogenously synthesized proteins. Binding to such peptides is a requirement for class I assembly in the endoplasmic reticulum (ER). A mutant human cell line, T2, assembles and transports to its surface some, but not all, class I MHC molecules. The class I molecules expressed on the surface of T2 do not present peptides derived from cytosolic antigens, although they can present exogenously added peptides to CTL. The transported class I molecules may interact weakly with an unknown retaining factor in the ER such that they can assemble despite the relative shortage of peptides.     

Structure, antigenic determinants of some clinically important insect allergens: chironomid hemoglobins

Determination of the molecular structure and properties of allergens that elicit severe immediate-type hypersensitivity diseases in humans and a knowledge of the structure of their antibody-binding sites should provide new insight into the pathogenetic mechanisms of allergic diseases. Monomeric and homodimeric hemoglobins (CTT I to X) have been identified as potent allergenic components of Chironomidae, a family of Diptera. Immunologic investigations of peptides of three of these hemoglobins (CTT IV, CTT VI, and CTT VIII) showed that human antibodies of the E and G classes recognize at least two different sites within each molecule. Individual hemoglobin peptides were aligned with homologous regions of chironomid hemoglobin CTT III, whose tertiary structure has been determined by x-ray analysis at a resolution of 1.4 angstroms. The antigenic site CTT IV(91 to 101) showed the following characteristics: (i) seven polar or hydroxylated amino acids, from a total of eleven, occupying predominantly superficial regions; (ii) the property of linkage to other molecules by hydrogen bonds or solvent clusters; and (iii) high thermal mobility factors. In contrast, peptide CTT IV(102 to 108), which does not bind human antibodies, contained no polar amino acids and had low thermal mobility factors. These results support the idea that the antigenicity of clinically relevant proteins is related to regions with a predominance of polar amino acids and with low energy barriers between different conformations, which allow high flexibility, including site-specific adaptation in antibody binding.     

Three-dimensional structure of poliovirus at 2.9 A resolution

The three-dimensional structure of poliovirus has been determined at 2.9 A resolution by x-ray crystallographic methods. Each of the three major capsid proteins (VP1, VP2, and VP3) contains a "core" consisting of an eight-stranded antiparallel beta barrel with two flanking helices. The arrangement of beta strands and helices is structurally similar and topologically identical to the folding pattern of the capsid proteins of several icosahedral plant viruses. In each of the major capsid proteins, the "connecting loops" and NH2- and COOH-terminal extensions are structurally dissimilar. The packing of the subunit "cores" to form the virion shell is reminiscent of the packing in the T = 3 plant viruses, but is significantly different in detail. Differences in the orientations of the subunits cause dissimilar contacts at protein-protein interfaces, and are also responsible for two major surface features of the poliovirion: prominent peaks at the fivefold and threefold axes of the particle. The positions and interactions of the NH2- and COOH-terminal strands of the capsid proteins have important implications for virion assembly. Several of the "connecting loops" and COOH-terminal strands form prominent radial projections which are the antigenic sites of the virion.     

Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells

In contrast to na?ve T cells that recognize short antigen-derived peptides displayed by specialized antigen-presenting cells, immunoglobulin receptors of B lymphocytes primarily recognize intact proteins. How and where within a lymph node such unprocessed antigens become available for na?ve B cell recognition is not clear. We used two-photon intravital imaging to show that, after exiting high-endothelial venules and before entry into lymph node follicles, B cells survey locally concentrated dendritic cells. Engagement of the B cell receptor by the dendritic cell (DC)-associated antigen leads to lymphocyte calcium signaling, migration arrest, antigen acquisition, and extrafollicular accumulation. These findings suggest a possible role for antigen-specific B-DC interactions in promoting T cell-dependent antibody responses in vivo.