Crystal structure of the Habc domain of neuronal syntaxin from the squid Loligo pealei reveals conformational plasticity at its C-terminus

BACKGROUND: Intracellular membrane fusion processes are mediated by the spatial and temporal control of SNARE complex assembly that results in the formation of a four-helical bundle, composed of one vesicle SNARE and three target membrane SNARE polypeptide chains. Syntaxins are essential t-SNAREs and are characterized by an N-terminal Habc domain, a flexible linker region, a coiled-coil or SNARE motif and a membrane anchor. The N-terminal Habc domain fulfills important regulatory functions while the coiled-coil motif, present in all SNAREs, is sufficient for SNARE complex formation, which is thought to drive membrane fusion. RESULTS: Here we report the crystal structure of the Habc domain of neuronal syntaxin from the squid Loligo pealei, s-syntaxin. Squid Habc crystallizes as a dimer and the monomer structure consists of a three-helical bundle. One molecule is strikingly similar to mammalian syntaxin 1A while the second one shows a structural deviation from the common fold in that the C-terminal part of helix C unwinds and adopts an extended conformation. CONCLUSION: Conservation of surface residues indicates that the cytosolic part of s-syntaxin can adopt an auto-inhibitory closed conformation that may bind squid neuronal Sec1, s-Sec1, in the same manner as observed in structure of the rat nSec1/syntaxin 1A complex. Furthermore, despite the overall structural similarity, the observed changes at the C-terminus of one molecule indicate structural plasticity in neuronal syntaxin. Implications of the structural conservation and the changes are discussed with respect to potential Habc domain binding partners such as Munc13, which facilitates the transition from the closed to the open conformation

Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction

We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the variable part of light and heavy chains of an antibody (Ab) from the chromosomal DNA of hybridoma cells for the chimerization ofAbs. A fundamental prerequisite for this is the knowledge ofthe exact sequences in the 5’-untranslated region of light and heavy chain mRNA, and of the joining segment used for rearrangement. This allows the design of nondegenerated oligodeoxyribonucleotides for PCR. The primer design permits directional cloning of the amplified, promoterless fragments into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin (Ig) promoter/enhancer elements. The method is illustrated for chimerization of an Ab directed against the human T-lymphocyte antigen, CD4. The chimerized Ab is secreted in abundant quantities after transfection of the engineered plasmids into non-Ig-producing myeloma cells

VH-RELATED IDIOTOPES DETECTED BY SITE-DIRECTED MUTAGENESIS

The function of the CD4 cell surface protein as coreceptor on T helper lymphocytes and as receptor for HIV makes this glycoprotein a prime target for an immune intervention with mAb. A detailed understanding of the structural determinants on the therapeutic CD4 mAb that are involved in Ag binding or are recognized by anti-idiotypic mAb (anti-Id) may be important for designing antibodies with optimal therapeutic efficacy. Seven anti-Id raised against the CD4 mAb M-T310 were selected from a large panel with the intention to obtain CD4 mimicking structures with specificity foHr IV gp120. The selected anti-Id did not reacwt ith other CDCspecific mAb cross-blocking M-T310. Among these, mAb MT404, although having the same L chain as M-T310 and a VH region sequence differing onlya t 14 amino acid positions, was not recognized by the anti-Id. MT310 H chain complexed with the J558L L chain reacted with all anti-Id, thus demonstrating that the recognized idiotopes are located within the VH region. To identify the idiotopes of M-T310 seen by the anti-Id, variants of M-T404 containing one or more of the M-T3 1 O-derived substitutions were generated by oligonucleotide-directed mutagenesis. The reactivity pattern of the mutant proteins with the anti-Id demonstrated that the idiotopes reside within the complementarity determining region (CDR) 2 and CDR3 loops of the VH region. A major idiotope was definebdy a single amino acid in CDR2 that was recognized by three anti-Id, whereas the four other anti-Id reacted with determinants of CDR3. Although the performed amino acid substitutions did influence the Id recognition, Ag binding was not significantly affected, suggesting that none of the anti-Id can be considered as a mimicry of the CD4 A

Crystal structure of subunit VPS25 of the endosomal trafficking complex ESCRT-II

BACKGROUND: Down-regulation of plasma membrane receptors via the endocytic pathway involves their monoubiquitylation, transport to endosomal membranes and eventual sorting into multi vesicular bodies (MVB) destined for lysosomal degradation. Successive assemblies of Endosomal Sorting Complexes Required for Transport (ESCRT-I, -II and III) largely mediate sorting of plasma membrane receptors at endosomal membranes, the formation of multivesicular bodies and their release into the endosomal lumen. In addition, the human ESCRT-II has been shown to form a complex with RNA polymerase II elongation factor ELL in order to exert transcriptional control activity. RESULTS: Here we report the crystal structure of Vps25 at 3.1 Å resolution. Vps25 crystallizes in a dimeric form and each monomer is composed of two winged helix domains arranged in tandem. Structural comparisons detect no conformational changes between unliganded Vps25 and Vps25 within the ESCRT-II complex composed of two Vps25 copies and one copy each of Vps22 and Vps36 [1,2]. CONCLUSIONS: Our structural analyses present a framework for studying Vps25 interactions with ESCRT-I and ESCRT-III partners. Winged helix domain containing proteins have been implicated in nucleic acid binding and it remains to be determined whether Vps25 has a similar activity which might play a role in the proposed transcriptional control exerted by Vps25 and/or the whole ESCRT-II complex

EXPRESSION OF A FUNCTIONAL CHIMERIC lg-MHC CLASS II PROTEIN

composed of the a- and ß-chains of the MHC class I1 I-E molecule fused to antibody V regions derived from anti-human CD4 mAb MT310. Expression vectors were constructed containing the functional, rearranged gene segments coding for the V region domains of the antibody H and L chains in place of the first domains of the complete structural genes of the I-E a- and ß-chains, respectively. Celltsr ansfected with both hybrid genes expressed a stable protein product on the cell surface. The chimeric molecule exhibited the idiotype of the antibody MT310 as shown by binding to the anti-idiotypic mAb 20-46. A protein of the anticipated molecular mass was immunoprecipitated witha nti-mouse IgG antiserum. Furthermore, human soluble CD4 did bind to thetr ansfected cell line, demonstrating that the chimeric protein possessed the binding capacity of the original mAb. Thus, the hybrid molecule retained: 1) the properties of a MHC class I1 protein with regardt o correct chain assembly and transport to the cell surface: as well as 2) the Ag binding capacity of the antibody genes used. Thgee neration of hybrid MHC class I1 molecules with highly specific, non-MHC-restricted bindingc apacities will be useful for studying MHC class 11-mediated effector functions such as selection of the T cell repertoire in thymus of transgenic mice

Combinatorial functions of two chimeric antibodies directed to human CD4 and one directed to the a-chain of the human interleukin-2 receptor

The general feasibility of chimerization of monoclonal antibodies (mAbs) has already been shown for a large number of them. In order to evaluate in vitro parameters relevant to immunosuppressive therapy, we have chimerized and synthesized two anti-CD4 mAbs recognizing two different epitopes on the human T-lymphocyte antigen, CD4. The chimerized mAbs are produced at levels corresponding to those of the original hybridoma cell lines. With respect to activation of human complement, the individual Abs are negative; however, when used in combination, complement activation was performed. When applied in combination, they were found to modulate the CD4 antigen, whereas the individual mAb do not display this property. Individually they mediate an up to 60% inhibition of the mixed lymphocyte reaction (MLR). However, by combination of an anti-CD4 mAb with one directed against the a-chain of the human IL2 receptor, nearly 100% inhibition of the MLR was achieved, even with reduced dosage of the mAbs. Our data suggest that the combination of an anti-CD4 mAb and an anti-IL2Rcc chain mAb is more effective with respect to immunosuppression than each mAb by itself, indicating that this mAb cocktail could be a new strategy for immunosuppressive therapy

Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction

We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the variable part of light and heavy chains of an antibody (Ab) from the chromosomal DNA of hybridoma cells for the chimerization ofAbs. A fundamental prerequisite for this is the knowledge ofthe exact sequences in the 5’-untranslated region of light and heavy chain mRNA, and of the joining segment used for rearrangement. This allows the design of nondegenerated oligodeoxyribonucleotides for PCR. The primer design permits directional cloning of the amplified, promoterless fragments into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin (Ig) promoter/enhancer elements. The method is illustrated for chimerization of an Ab directed against the human T-lymphocyte antigen, CD4. The chimerized Ab is secreted in abundant quantities after transfection of the engineered plasmids into non-Ig-producing myeloma cells

Роль метода электрофоретического осаждения в создании биокомпозита на основе слоев гидроксиапатити и наночастиц серебра

Работа посвящена созданию многофункционального биокомпозита, состоящего из покрытия на основе гидроксиапатита (ГА) и наночастиц серебра с использованием высокотехнологичных методов обработки поверхности. Высокочастотное магнетронное распыление использовалось для получения слоев ГА покрытия с различной толщиной и структурой на титане и наночастицах серебра. Для получения антибактериального слоя наночастиц серебра использовался метод электрофоретического осаждения. Наночастицы серебра имели сферическую форму с диаметром 70±20 нм и[zeta] -потенциалом -20 мВ. Дифракционные картины биокомпозитов выявили пики кристаллического ГА и серебра (Ag). Так же установлено, что наночастицы серебра являются кристаллическими с размером кристаллитов 14 нм

Cryo-Electron Microscopy Three-Dimensional Structure of the Jumbo Phage ΦRSL1 Infecting the Phytopathogen Ralstonia solanacearum

SummaryϕRSL1 jumbo phage belongs to a new class of viruses within the Myoviridae family. Here, we report its three-dimensional structure determined by electron cryo microscopy. The icosahedral capsid, the tail helical portion, and the complete tail appendage were reconstructed separately to resolutions of 9 Å, 9 Å, and 28 Å, respectively. The head is rather complex and formed by at least five different proteins, whereas the major capsid proteins resemble those from HK97, despite low sequence conservation. The helical tail structure demonstrates its close relationship to T4 sheath proteins and provides evidence for an evolutionary link of the inner tail tube to the bacterial type VI secretion apparatus. Long fibers extend from the collar region, and their length is consistent with reaching the host cell surface upon tail contraction. Our structural analyses indicate that ϕRSL1 is an unusual member of the Myoviridae that employs conserved protein machines related to different phages and bacteria