The Structural Basis for Specific Recognition of H3K14 Acetylation by Sth1 in the RSC Chromatin Remodeling Complex

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Structural Insights into the Substrate Specificity Switch Mechanism of the Type III Protein Export Apparatus

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Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.     

快速展示糖苷水解酶活性架构单点突变导致结合力变化的电泳技术

酶分子在长期进化过程中形成一系列氨基酸残基组成的活性架构,参与底物的识别、结合与催化过程,而活性架构中相应氨基酸残基是如何影响酶分子结合底物的能力,进而影响酶分子的催化效率,一直是酶分子理性改造研究的热点.利用亲和电泳技术,可以快速展示内切纤维素酶Tr Cel12A和木聚糖酶Tl Xyn A活性架构中不同突变体的催化活性及其迁移率的变化,进而通过在不同底物浓度凝胶中蛋白质相对迁移率变化程度的定量回归分析,发现由氨基酸单点突变导致蛋白质迁移率的相对变化,可以定量表征酶分子突变前后结合底物能力的变化.亲和电泳测定的有效阻滞常数Kb值与等温滴定量热法和荧光光谱法测定的相关参数比较具有明显相关性.由于亲和电泳技术在测定酶分子与底物的结合能力时具有简便、快速、灵敏的特点,因而可作为常规生化实验室常规普筛技术来检测突变文库中系列突变体导致结合力的变化.     

The Substrate-free and -bound Crystal Structures of the Duplicated Taurocyamine Kinase from the Human Parasite Schistosoma mansoni

The taurocyamine kinase from the blood fluke Schistosoma mansoni (SmTK) belongs to the phosphagen kinase (PK) family and catalyzes the reversible Mg²⁺-dependent transfer of a phosphoryl group between ATP and taurocyamine. SmTK is derived from gene duplication, as are all known trematode TKs. Our crystallographic study of SmTK reveals the first atomic structure of both a TK and a PK with a bilobal structure. The two unliganded lobes present a canonical open conformation and interact via their respective C- and N-terminal domains at a helix-mediated interface. This spatial arrangement differs from that observed in true dimeric PKs, in which both N-terminal domains make contact. Our structures of SmTK complexed with taurocyamine or l-arginine compounds explain the mechanism by which an arginine residue of the phosphagen specificity loop is crucial for substrate specificity. An SmTK crystal was soaked with the dead end transition state analog (TSA) components taurocyamine-NO₃²⁻-MgADP. One SmTK monomer was observed with two bound TSAs and an asymmetric conformation, with the first lobe semiclosed and the second closed. However, isothermal titration calorimetry and enzyme kinetics experiments showed that the two lobes function independently. A small angle x-ray scattering model of SmTK-TSA in solution with two closed active sites was generated.     

Structure of the UHRF1 Tandem Tudor Domain Bound to a Methylated Non-histone Protein,LIG1, Reveals Rules for Binding and Regulation

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VLR Recognition of TLR5 Expands the Molecular Characterization of Protein Antigen Binding by Non-Ig-based Antibodies

Variable lymphocyte receptors (VLRs) are unconventional adaptive immune receptors relatively recently discovered in the phylogenetically ancient jawless vertebrates, lamprey and hagfish. VLRs bind antigens using a leucine-rich repeat fold and are the only known adaptive immune receptors that do not utilize an immunoglobulin fold for antigen recognition. While immunoglobulin antibodies have been studied extensively, there are comparatively few studies on antigen recognition by VLRs, particularly for protein antigens. Here we report isolation, functional and structural characterization of three VLRs that bind the protein toll-like receptor 5 (TLR5) from zebrafish. Two of the VLRs block binding of TLR5 to its cognate ligand flagellin in functional assays using reporter cells. Co-crystal structures revealed that these VLRs bind to two different epitopes on TLR5, both of which include regions involved in flagellin binding. Our work here demonstrates that the lamprey adaptive immune system can be used to generate high-affinity VLR clones that recognize different epitopes and differentially impact natural ligand binding to a protein antigen.