Cloning, purification and preliminary crystallographic analysis of a putative pyridoxal kinase from Bacillus subtilis

A putative pyridoxal kinase from B. subtilis has been cloned, overexpressed, purified and crystallized and data have been collected to 2.8 Å resolution

Improving Factuality of Abstractive Summarization via Contrastive Reward Learning

Modern abstractive summarization models often generate summaries that contain hallucinated or contradictory information. In this paper, we propose a simple but effective contrastive learning framework that incorporates recent developments in reward learning and factuality metrics. Empirical studies demonstrate that the proposed framework enables summarization models to learn from feedback of factuality metrics using contrastive reward learning, leading to more factual summaries by human evaluations. This suggests that further advances in learning and evaluation algorithms can feed directly into providing more factual summaries.Comment: TrustNLP @ ACL 202

Structural Comparison of Human Mammalian Ste20-Like Kinases

BACKGROUND: The serine/threonine mammalian Ste-20 like kinases (MSTs) are key regulators of apoptosis, cellular proliferation as well as polarization. Deregulation of MSTs has been associated with disease progression in prostate and colorectal cancer. The four human MSTs are regulated differently by C-terminal regions flanking the catalytic domains. PRINCIPAL FINDINGS: We have determined the crystal structure of kinase domain of MST4 in complex with an ATP-mimetic inhibitor. This is the first structure of an inactive conformation of a member of the MST kinase family. Comparison with active structures of MST3 and MST1 revealed a dimeric association of MST4 suggesting an activation loop exchanged mechanism of MST4 auto-activation. Together with a homology model of MST2 we provide a comparative analysis of the kinase domains for all four members of the human MST family. SIGNIFICANCE: The comparative analysis identified new structural features in the MST ATP binding pocket and has also defined the mechanism for autophosphorylation. Both structural features may be further explored for inhibitors design. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

Application of Structural Genomics for Developing Novel Antibacterial Agents

The structure-based design of novel antibiotics has great potentiaL To achieve this goal the first step is to gather structural information on relevant proteins and utilise this to find inhibitors through a process of screening and subsequent optimising using structural data. In this· pilot study a number of essential gene products from Bacillus subtilis were targeted for structural studies where homologues were present in pathogenic microorganisms. In the initial analysis 41 essential genes of unknown structure or function were selected for structure determination. From this list 9 genes were rejected as their protein products were assigned as possible membrane proteins by hydropathy analysis. From the remaining 32 genes on the list 12 (ysxC, thiD, ywaF, ydiC, ydiE, yrrA, yabH, ylxR, yqiB, yacN, ymjL and luxS) were selected for study as part of this thesis. Recombinant DNA technology was used to produce pure protein for crystallisation trials and subsequent structure determination by X-ray crystallography. Successful overexpression was achieved for 8 of the protein targets ofwhich 7 were found to be soluble. 6 ofthese were taken forward for crystallisation as part of this work with the 7th being·pursued by another member of the laboratory due to project overlap. Of these 6 proteins 5 were successfully purified and crystallised (LuxS, YsxC, YacN, YabH, and ThiD) and structures were determined for all but ThiD. The successful crystallisation of all of the 5 soluble proteins was unexpected and maybe related to the characteristics of the structure determination pipeline which requires the expression of the protein product in a soluble form and at high leveL This may result in the selection of proteins with a higher tendency to crystallise. The gene to structure conversion rate'obtained here (33%) is equivalent to that obtained for the overall programme on all 32 genes (34%) suggesting that 4 bacterial proteins 1/3rd of the proteome can be analysed as part of a structural genomics programme.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Kinase Domain Insertions Define Distinct Roles of CLK Kinases in SR Protein Phosphorylation

Splicing requires reversible phosphorylation of serine/arginine-rich (SR) proteins, which direct splice site selection in eukaryotic mRNA. These phosphorylation events are dependent on SR protein (SRPK) and cdc2-like kinase (CLK) families. SRPK1 phosphorylation of splicing factors is restricted by a specific docking interaction whereas CLK activity is less constrained. To understand functional differences between splicing factor targeting kinases, we determined crystal structures of CLK1 and CLK3. Intriguingly, in CLKs the SRPK1 docking site is blocked by insertion of a previously unseen helix αH. In addition, substrate docking grooves present in related mitogen activating protein kinases (MAPKs) are inaccessible due to a CLK specific β7/8-hairpin insert. Thus, the unconstrained substrate interaction together with the determined active-site mediated substrate specificity allows CLKs to complete the functionally important hyperphosphorylation of splicing factors like ASF/SF2. In addition, despite high sequence conservation, we identified inhibitors with surprising isoform specificity for CLK1 over CLK3