Yongxiang Gao

Core Structure of the Yeast Spt4-Spt5 Complex: A Conserved Module for Regulation of Transcription Elongation

The Spt4-Spt5 complex is an essential RNA polymerase II elongation factor found in all eukaryotes and important for gene regulation. We report here the crystal structure of Saccharomyces cerevisiae Spt4 bound to the NGN domain of Spt5. This structure reveals that Spt4-Spt5 binding is governed by an acid-dipole interaction between Spt5 and Spt4. Mutations that disrupt this interaction disrupt the complex. Residues forming this pivotal interaction are conserved in the archaeal homologs of Spt4 and Spt5, which we show also form a complex. Even though bacteria lack a Spt4 homolog, the NGN domains of Spt5 and its bacterial homologs are structurally similar. Spt4 is located at a position that may help to maintain the functional conformation of the following KOW domains in Spt5. This structural and evolutionary perspective of the Spt4-Spt5 complex and its homologs suggest that it is an ancient, core component of the transcription elongation machinery.

Structural basis of pre-mRNA recognition by the human cleavage factor Im complex

The cleavage factor Im (CF Im), consists of a 25 kDa subunit (CF Im25) and one of three larger subunits (CF Im59, CF Im68, CF Im72), and is an essential protein complex for pre-mRNA 3′-end cleavage and polyadenylation. It recognizes the upstream sequence of the poly(A) site in a sequence-dependent manner. Here we report the crystal structure of human CF Im, comprising CF Im25 and the RNA recognition motif domain of CF Im68 (CF Im68RRM), and the crystal structure of the CF Im-RNA complex. These structures show that two CF Im68RRM molecules bind to the CF Im25 dimer via a novel RRM-protein interaction mode forming a heterotetramer. The RNA-bound structure shows that two UGUAA RNA sequences, with anti-parallel orientation, bind to one CF Im25-CF Im68RRM heterotetramer, providing structural basis for the mechanism by which CF Im binds two UGUAA elements within one molecule of pre-mRNA simultaneously. Point mutation and kinetic analyses demonstrate that CF Im68RRM can bind the immediately flanking upstream region of the UGUAA element, and CF Im68RRM binding significantly increases the RNA-binding affinity of the complex, suggesting that CF Im68 makes an essential contribution to pre-mRNA binding.

Crystal structure and possible dimerization of the single RRM of human PABPN1

Crystal structure and possible dimerization of the single RRM of human PABPN1 Honghua Ge, Dongwen Zhou, Shuilong Tong, Yongxiang Gao, Maikun Teng,* and Liwen Niu* 1Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China 2 Key Laboratory of Structural Biology, Chinese Academy of Sciences, Hefei, Anhui 230026, China 3Modern Experiment Technology Center, Anhui University, Hefei, Anhui 230039, China

Structure of native laccase B from Trametes sp. AH28-2.

Fungal laccases are oxidoreductases that belong to the multinuclear copper-containing oxidases. They are able to oxidize a wide range of substrates, preferably phenolic compounds, which makes them suitable for employment in the bioremediation of soil and water as well as in other biotechnological applications. Here, the structural analysis of natural laccase B (LacB) from Trametes sp. AH28-2 is presented. This structure provides the opportunity to study the natural post-translational modifications of the enzyme. The overall fold shows a high homology to those of previously analyzed laccases with known three-dimensional structure. However, LacB contains a new structural element, a protruding loop near the substrate-binding site, compared with the previously reported laccase structures. This unique structural feature may be involved in modulation of the substrate recognition of LacB.

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.

Crystal structure of the C-terminal conserved domain of human GRP, a galectin-related protein, reveals a function mode different from those of galectins.

Lectins are a group of proteins that recognize carbohydrates covalently linked to proteins and lipids on the cell surface and within the extracellular matrix and have diverse physiological functions1–3 including growth regulation,4–6 cell adhesion,7,8 pre-mRNA splicing,9,10 cell migration,11,12 cell apoptosis,13–15 immune responses,16 and pathogen recognition.17 The galectins are a family of animal lectins defined by their shared conserved carbohydrate recognition domain (CRD) of about 130 amino acids and affinity for b-galactoside sugars.18,19 The galectins can be classified into three subfamilies as proto-, chimera-, and tandem-repeat types based on their domain organization.2,20 Gal-1, 2, 5, 7, 10, 11, 13, 14, and 15 are prototype galectins which contain one carbohydrate-recognition domain per subunit and are usually homodimers of noncovalently linked subunits.3,21 Gal3 is the only member of the chimera-type galectin which contains a C-terminal CRD and an N-terminal slightly long peptide rich in praline and glycine.22 Gal4, 6, 8, 9, and 12 are examples of tandem-repeat galectins which contain two CRDs joined by a linked peptide and are monomeric.23 Fifteen mammalian galectins have been described so far.21,24 Meanwhile, several galectin relatives such as lens crystalline protein GRIFIN and the hematopoietic stem cell precursor, HSPC159 have become available.24 The same property of these proteins is the significant sequence deviations at the most critical residues for carbohydrate-binding, leading to that they all lack b-galactosides binding activity. In the past years, the X-ray crystal structures of a few galectins such as gal-1,25–27 2,28,29 3,30 7,31 and 1032 have been reported and they are all similar and show jelly-roll topologies typical of legume lectins. Their CRDs are all composed of 11 or 12-strand antiparallel b-sandwich. Some of them have short 310 helices. The general architectures of the carbohydrate-binding site in galectins of known three dimension structures are very similar. The structure of human Gal-1-b-galactoside complex reveals that the amino acids His44, Asn46, Arg48, Val59,

Crystal structure of NusG N‐terminal (NGN) domain from Methanocaldococcus jannaschii and its interaction with rpoE″

Transcription in archaea employs a eukaryotic‐type transcription apparatus but uses bacterial‐type transcription factors. NusG is one of the few archaeal transcription factors whose orthologs are essential in both bacteria and eukaryotes. Archaeal NusG is composed of only an NusG N‐terminal (NGN) domain and a KOW domain, which is similar to bacterial NusG but not to the eukaryotic ortholog, Spt5. However, archaeal NusG was confirmed recently to form a complex with rpoE″ that was similar to the Spt5‐Spt4 complex. Thus, archaeal NusG presents hybrid features of Spt5 and bacterial NusG. Here we report the crystal structure of NGN from the archaea Methanocaldococcus jannaschii (MjNGN). MjNGN folds to an α‐β‐α sandwich without the appendant domain of bacterial NGNs, and forms a unique homodimer in crystal and solution. MjNGN alone was found to be sufficient for rpoE″ binding and an MjNGN‐rpoE″ model has been constructed by rigid docking. Proteins 2009.

Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris

l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris (pvLAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pvLAAD does not resemble typical LAAOs. Instead it, is similar to d-amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pvLAAD could generate a significant amount of hydrogen peroxide in vitro, suggesting it could use dioxygen to directly re-oxidize FADH2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications.

Structures of enzyme-intermediate complexes of yeast Nit2: insights into its catalytic mechanism and different substrate specificity compared with mammalian Nit2

The Nit (nitrilase-like) protein subfamily constitutes branch 10 of the nitrilase superfamily. Nit proteins are widely distributed in nature. Mammals possess two members of the Nit subfamily, namely Nit1 and Nit2. Based on sequence similarity, yeast Nit2 (yNit2) is a homologue of mouse Nit1, a tumour-suppressor protein whose substrate specificity is not yet known. Previous studies have shown that mammalian Nit2 (also a putative tumour suppressor) is identical to ω-amidase, an enzyme that catalyzes the hydrolysis of α-ketoglutaramate (α-KGM) and α-ketosuccinamate (α-KSM) to α-ketoglutarate (α-KG) and oxaloacetate (OA), respectively. In the present study, crystal structures of wild-type (WT) yNit2 and of WT yNit2 in complex with α-KG and with OA were determined. In addition, the crystal structure of the C169S mutant of yNit2 (yNit2-C169S) in complex with an endogenous molecule of unknown structure was also solved. Analysis of the structures revealed that α-KG and OA are covalently bound to Cys169 by the formation of a thioester bond between the sulfhydryl group of the cysteine residue and the γ-carboxyl group of α-KG or the β-carboxyl group of OA, reflecting the presumed reaction intermediates. However, an enzymatic assay suggests that α-KGM is a relatively poor substrate of yNit2. Finally, a ligand was found in the active site of yNit2-C169S that may be a natural substrate of yNit2 or an endogenous regulator of enzyme activity. These crystallographic analyses provide information on the mode of substrate/ligand binding at the active site of yNit2 and insights into the catalytic mechanism. These findings suggest that yNit2 may have broad biological roles in yeast, especially in regard to nitrogen homeostasis, and provide a framework for the elucidation of the substrate specificity and biological role of mammalian Nit1.

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.