Zhiqiang Zhu

Crystal structure of human vacuolar protein sorting protein 29 reveals a phosphodiesterase/nuclease-like fold and two protein-protein interaction sites

Vacuolar protein sorting protein 29 (Vps29p), which is involved in retrograde trafficking from prevacuolar endosomes to the trans-Golgi network, performs its biological functions by participating in the formation of a “retromer complex.” In human cells, this complex comprises four conserved proteins: hVps35p, hVps29p, hVps26p, and sorting nexin 1 protein (SNX1). Here, we report the crystal structure of hVps29p at 2.1 Å resolution, the first three-dimensional structure of the retromer subunits. This novel structure adopts a four-layered α-β-β-α sandwich fold. hVps29p contains a metal-binding site that is very similar to the active sites of some proteins of the phosphodiesterase/nuclease protein family, indicating that hVps29p may carry out chemically similar functions. Structure and sequence conservation analysis suggests that hVps29p contains two protein-protein interaction sites. One site, which potentially serves as the interface between hVps29p and hVps35p, comprises 5 conserved hydrophobic and 8 hydrophilic residues. The other site is relatively more hydrophilic and may serve as a binding interface with hVps26p, SNX1, or other target proteins.

Epitope mapping and structural analysis of an anti-ErbB2 antibody A21: Molecular basis for tumor inhibitory mechanism

Anti‐ErbB2 antibodies targeting distinct epitopes can have different biological functions on cancer cells. A21 prepared by surface epitope masking (SEM) method is a tumor‐inhibitory anti‐ErbB2 monoclonal antibody. Previously we engineered a single chain chimeric antibody chA21 with potential for therapy of ErbB2‐overexpressing tumors. Here, we mapped the A21 epitope on ErbB2 extracellular domain (ECD) by screening a combinatorial phage display peptide library, serial subdomain deletion, and mutagenesis scanning. X‐ray crystal structure of the A21 scFv fragment at 2.1 Å resolution was also determined. A molecular model of Ag‐Ab complex was then constructed based on the crystal structures of the A21 scFv and ErbB2 ECD. Some of biological functions of the A21 mAb and its derivative antibodies including their tumor cell growth inhibition and effects on the expression, internalization, and phosphorylation of ErbB2 receptor were also investigated. The results showed that A21 recognized a conformational epitope comprising a large region mostly from ErbB2 extracellular subdomain I with several surface‐exposed residues important for the binding affinity. These data provide unique functional properties of A21 that are quite different from two broadly used anti‐ErbB2 mAbs, Herceptin and 2C4. It suggested that the A21 epitope may be another valuable target for designing new anti‐ErbB2 therapeutics. Proteins 2008.

The crystal structure and identification of NQM1/YGR043C, a transaldolase from Saccharomyces cerevisiae

Transaldolase belongs to the class I aldolase family, whose members are the pivotal enzymes catalyzing the most important carbon-carbon bond-forming reactions and aldol condensation reactions.1 Different from the members of the class II adolase family, transaldolase is characterized by the formation of a covalent Schiff base intermediate between a lysine residue within the active site and the substrate.2–4 As a key enzyme in the pentose phosphate pathway, transaldolase catalyzes the reversible transfer of a dihydroxyacetone moiety from fructose 6-phosphate to erythrose 4-phosphate and forms sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate, respectively.4–6 The vital functions of this enzyme are reported to balance the levels of NADPH and reactive oxygen intermediates (ROI) in the pentose phosphate pathway of glucose metabolism7 and also to maintain the mitochondrial transmembrane potential and sperm fertility.8 Dysfunction of transaldolase attracts a great deal of attention since it could lead to several severe diseases, such as liver cirrhosis9 and neonatal multi-organ disease.10 In the Saccharomyces cerevisiae genome, one open reading frame, named NQM1/YGR043C, exhibits more than 50% sequence identity to a group of transaldolases both from prokaryotic and eukaryotic organisms, such as Escherichia coli transaldolase B (TALB_ECOLI), Mus musculus transaldolase (TAL_MOUSE), and Homo sapiens transaldolase (TAL_HUMAN). According to the sequence alignment, NQM1/YGR043C was proposed to be a putative transaldolase in yeast (TAL2_YEAST).11 However, no experimental evidence has been reported to prove its transaldolase activity to date. In our studies, the NQM1/YGR043C gene from the cDNA library of S. cerevisiae was cloned into a pET22b(1) vector and expressed in Escherichia coli strain BL21 (DE3). The purified protein was crystallized using the hanging-drop vapor diffusion method and the crystal structure was determined by the molecular replacement method and refined to a 1.9 Å resolution. The NQM1/ YGR043C protein folds into an eight-stranded a/b barrel, which is conserved among all reported transaldolase structures. The enzyme activity assays further demonstrated its transaldolase activity. Based on these data, we concluded that the open reading frame (ORF) of NQM1/ YGR043C does encode a transaldolase, which could be named as TAL2_YEAST.

Structural basis of the autolysis of AaHIV suggests a novel target recognizing model for ADAM/reprolysin family proteins.

AaHIV, a P-III-type snake venom metalloproteinase (SVMP), consists of metalloproteinase/disintegrin/cysteine-rich (MDC) domains and is homologous to a disintegrin and metalloproteinase (ADAM) family proteins. Similar to brevilysin H6 and jararhagin, AaHIV can easily autolyse to release a stable protein named acucetin, which contains disintegrin-like and cysteine-rich domains. In this study, we determined the crystal structure of AaHIV and investigated the autolysis mechanism. Based on the structure of AaHIV and the results from docking experiments, we present a new model for target recognition in which two protein molecules form a functional unit, and the DC domain of one molecule is used for target recognition while the M-domain of the other is used for target proteolysis. Our results shed new light on the mechanism of target recognition and processing in ADAM/reprolysin family proteins.

Purification, partial characterization and crystallization of acucetin, a protein containing both disintegrin-like and cysteine-rich domains released by auto-proteolysis of a P-III-type metalloproteinase AaH-IV from Agkistrodon acutus venom.

AaH-IV, a P-III-type metalloproteinase found in Agkistrodon acutus venom, readily cleaves itself to release a stable protein named acucetin at 310 K under neutral and weakly alkaline conditions. A partial amino-acid residue sequence of acucetin indicates that the protein has a high homology to snake-venom proteins containing both disintegrin-like and cysteine-rich domains. Acucetin has been crystallized in space group R32, with hexagonal unit-cell parameters a = b = 155.98, c = 76.07 A. The V(M) value of about 2.97 A(3) Da(-1) suggests the presence of only one molecule in the asymmetric unit.

Stejnihagin, a novel snake metalloproteinase from Trimeresurus stejnegeri venom, inhibited L-type Ca2+ channels

Snake venom metalloproteinases (SVMPs) mainly distribute in Crotalid and Viperid snake venom and are classified into the Reprolysin subfamily of the M12 family of metalloproteinases. Previous function investigations have suggested that SVMPs are the key toxins involved in a variety of snake venom-induced pathogenesis including systemic injury, local damage, hemorrhage, edema, hypotension, hypovolemia, inflammation and necrosis. However, up to now, there is no report on ion channels blocking activity about SVMPs. Here, from Trimeresurus stejnegeri venom we purified a component Stejnihagin containing a mixture of Stejnihagin-A and -B, with 86% sequences identity, both as members of SVMPs. In the study, whole-cell patch clamp and vessel tension measurement were employed to identify the effect of Stejnihagin on L-type Ca2+ channels and vessel contraction. The results show that Stejnihagin inhibited L-type Ca2+ channels in A7r5 cells with an IC50 about 37 nM and simultaneously blocked 60 mM K+-induced vessel contraction. Besides, the inhibitory effect of Stejnihagin on L-type Ca2+ channels was also independent of the enzymatic activity. This finding offers new insight into the snake venom metalloproteinase functions and provides a novel pathogenesis of T. stejnegeri venom. Furthermore, it may also provide a clue to study the structure-function relationship of animal toxins and voltage-gated Ca2+ channel.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of human phosphoribosyl pyrophosphate synthetase 1 (PRS1)

Phosphoribosyl pyrophosphate synthetase (PRS; EC 2.7.6.1) catalyzes the reaction of ribose-5-phosphate (R5P) with ATP to yield AMP and PRPP (5-phosphoribosyl-1-pyrophosphate), which is necessary for the de novo and salvage pathways of purine-, pyrimidine- and pyridine-nucleotide biosynthesis. PRPP is a metabolite that is required at all times in the cell and is thus central to life. In this study, human PRS1 was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals in complex with Mg2+, inorganic phosphate (P(i)) and ATP were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.6 A resolution. The crystal belongs to space group R3, with unit-cell parameters a = b = 168.846, c = 61.857 angstroms, assuming two molecules in the asymmetric unit and a volume-to-weight ratio of 2.4 angstroms3 Da(-1), which was consistent with the result calculated from the self-rotation function.

Crystal structure of the two N-terminal RRM domains of Pub1 and the poly(U)-binding properties of Pub1

Yeast poly(U)-binding protein (Pub1) is a major nuclear and cytoplasmic protein that contains three RNA recognition motif (RRM) domains (termed Pub1RRM1, Pub1RRM2 and Pub1RRM3). Pub1 has been implicated as a regulator of cellular mRNA decay. Nearly 10% of all yeast mRNA decay occurs in a Pub1-dependent manner. Pub1 binds to and stabilizes AU-rich element (ARE) and ARE-like sequence-containing transcripts by protecting them from degradation through the deadenylation-dependent pathway, and also binds to and stabilizes stabilizer element (STE)-containing transcripts by preventing their degradation via the nonsense-mediated decay (NMD) pathway. RNA-binding analyses showed that Pub1 binds to poly(U) in vitro. Here we show the crystal structures of Pub1RRM2 and the first two tandem RRM domains (Pub1RRM12). Crystallography showed that the structure of Pub1RRM12 is a domain-swapped dimer. Size exclusion chromatography assay and analytical ultracentrifugation (AUC) showed that Pub1RRM12 is a monomer in solution. Kinetic analysis showed that all three individual RRM domains can bind to poly(U) with similar affinities and Pub1RRM12 binds to a long poly(U) segment with higher affinity. Mutagenesis analysis revealed that residues on the beta-sheets of Pub1RRM1 and Pub1RRM2 are critical for poly(U) binding.

Crystal structure of human osteoclast stimulating factor.

Osteoclast is the main cell responsible for the degradation of bone matrix.1 Its differentiation and activity requires a variety of factors. For example, macrophage colony stimulating factor (M-CSF) prevents apoptosis of early osteoclast precursors,2 and receptor activator of nuclear factor jB ligand (RANKL) is very important for osteoclast formation and bone remodeling.3,4 Tyrosine protein kinase Src, involved in both M-CSF and RANKL mediated signal networks,4,5 was also found to be essential for osteoclast function.6,7 In addition, factors produced by osteoclasts themselves (such as interleukin-6, transforming growth factor beta, etc.) also play important roles.8–10 Osteoclast stimulating factor (OSF), composed of a proline-rich region, a SH3 domain and ankyrin repeats, is a intracellular protein produced by osteoclasts and shown to indirectly enhance osteoclast formation and activity.11 A mouse homolog of OSF called SH3P2 (95% sequence identity with OSF) can bind proline-rich fragment of Cbl and form an OSF-Cbl-Src triple protein complex,12 suggesting that OSF was involved in Srcand Cblmediated pathways. OSF-SH3 domain was also found to bind exon 6 region of survival motor neuron (a product of spinal muscular atrophy disease-determining gene).13 SH3 domains recognize proline-rich sequences with a PxxP motif, which was further classified into 1xuPxuP (class I) and uPxuPx1 (class II)14,15 (where x is any amino acid, u is usually a hydrophobic residue, and 1 is usually an arginine residue). This motif adopts a lefthanded polyproline type II helical conformation,14 known as a collagen chain conformation, and fits the ligand-binding groove on the SH3 surface. Ankyrin repeats are common protein scaffolds and have been found in many proteins spanning a wide range of functions.16 They mediate protein–protein interactions mostly via a concave inner surface formed by a loop region and inner short helices through hydrophilic contacts, such as hydrogen bonds and salt bridges.17 Here, we present crystal structures of human OSF in two space groups grown from the same condition. One is determined by multiwavelength anomalous diffraction (MAD) at 2.57 Å resolution in space group P212121, and the other is determined by molecular replacement at 1.95 Å resolution in space group P1. Models in both crystal forms contain one SH3 domain and four ankyrin repeats, but the relative position of the two domains is different in two forms. It is the first reported protein structure of such a domain sequence. A prolonged and more standard helix was found to be unique in OSF-SH3 domain, and residues that may be responsible for the binding specificity of OSF ankyrin repeats were also discussed.

Structure of the second PDZ domain from human zonula occludens 2

Human zonula occludens 2 (ZO-2) protein is a multi-domain protein that consists of an SH3 domain, a GK domain and three copies of a PDZ domain with slight divergence. The three PDZ domains act as protein-recognition modules that may mediate protein assembly and subunit localization. The crystal structure of the second PDZ domain of ZO-2 (ZO-2 PDZ2) was determined by molecular replacement at 1.75 A resolution, revealing a dimer in the asymmetric unit. The dimer is stabilized by extensive symmetrical domain-swapping of the beta1 and beta2 strands. Structural comparison shows that the ZO-2 PDZ2 homodimer may have a similar ligand-binding pattern to the ZO-1 PDZ2-connexin 43 complex.