分类： 生物学 >> 生物物理学 提交时间： 2016-05-12
摘要： Tandem duplications and fusions of single genes have led to magnificent expansions in the divergence of protein structures and functions over evolutionary timescales. One of the possible results is polydomain enzymes with interdomain cooperativities, few examples of which have been structurally characterized at the full-length level to explore their innate synergistic mechanisms. This work reports the crystal structures of a double-domain phosphagen kinase in both apo and ligand-bound states, revealing a novel asymmetric L-shaped arrangement of the two domains. Unexpectedly, the interdomain connections are not based on a flexible hinge linker but on a rigid secondary-structure element: a long alpha-helix that tethers the tandem domains in relatively fixed positions. Besides the connective helix, the two domains also contact each other directly and form an interdomain interface in which hydrogen bonds and hydrophobic interactions further stabilize the L-shaped domain arrangement. Molecular-dynamics simulations show that the interface is generally stable, suggesting that the asymmetric domain arrangement crystallographically observed in the present study is not a conformational state simply restrained by crystal-packing forces. It is possible that the asymmetrically arranged tandem domains could provide a structural basis for further studies of the interdomain synergy.
分类： 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间： 2016-05-12
摘要： Talin, as the activator of integrin and the adaptor between the cytoskeleton and integrin, plays a key role in a series of processes such as cell adhesion and migration. The activation of integrin involves F3 subdomain of Talin-FERM domain binding the cytoplasmic tail of integrin beta-subunit. Talin has two states: auto-inhibited and activated. We previously reported the auto-inhibition complex structure of Talin F2F3/R9, in which the integrin binding site F3 interacts with R9(1654 similar to 1822 a.a.) of Talin-ROD, such that integrin cannot be activated. However, besides F3 and R9, it remains unclear what structural or functional roles the other domains of the 270 ku Talin play in the regulation of its activation. Here we solved the crystal structures of Talin R9-R10 (1654 similar to 1973 a.a.) and R10-R11 (1815 similar to 2140 a.a.), respectively. R9, R10 and R11 are all 5-helix bundles. R9 and R10 is joined together by a long alpha-helix instead of a flexible loop, and the two bundles are located at the opposite sides of the long helix with an angle of about 150 degrees. The linker between R10 and R11 is stabilized by neighboring hydrogen bonds, forming an angle of about 120 between the two bundles. These angles observed in our crystal structures are consistent with the previously reported SAXS and EM results. After superimposition of R9-10, R10-11 with previously reported structures of R7-8 and R11-12, a model of R7-12 was acquired, which adopts an elongated linear conformation, except that R8 protrudes from the ROD. According to this model, R10-12 does not intrude the interaction between F3 and R9, whereas R8 not only masks the F3 binding site of R9, but also might electrostatically hinders F2F3 approaching via its unique positively charged surface. This hypothesis was further verified by the results of size exclusion chromatography. Our work provides a new structural basis for studying the mechanism of Talin auto-inhibition.
分类： 生物学 >> 生物物理学 >> 生物力学与生物流变学 提交时间： 2016-05-12
Zeng Yan
Zhang Yong
Ji QingHua
Lou JiZhong
Song XianQiang
Ye Sheng
Zhang RongGuang
Zeng Yan
Song XianQiang
Ji QingHua
摘要： Talin is an integrin-binding protein located at focal adhesion site and serves as both an adapter and a force transmitter. Its integrin binding activity is regulated by the intramolecular autoinhibition interaction between its F3 and RS domains. Here, we used atomic force microscopy to measure the strength of talin autoinhibition complex. Our results suggest that the lifetime of talin autoinhibition complex shows weak catch bond behavior and does not change significantly at smaller forces, while it drops rapidly at larger forces (>10 pN). Moreover, besides the complex conformation revealed by crystal structure, our molecular dynamics (MD) simulations indicate the possible existence of another stable conformation. Further analysis indicates that forces may regulate the equilibrium of the two stable binding states and result in the non-exponential force dependence of the binding lifetime. Our findings reveal a negative regulation mechanism on talin activation and provide a new point of view on the function of talin in focal adhesion.
分类： 生物学 >> 生物物理学 提交时间： 2016-05-11
Zhu, Xiaojie
Wang, Qian
Xu, Wei
Su, Shan
Sun, Zhiwu
Yu, Fei
Liu, Qi
Zhang, Xiaoyan
Xu, Jianqing
Lu, Lu
Jiang, Shibo
Zhu, Xiaojie
Wang, Qian
Xu, Wei
Su, Shan
Sun, Zhiwu
Yu, Fei
Liu, Qi
Zhang, Xiaoyan
Xu, Jianqing
摘要： Enfuvirtide (T20), is the first HIV fusion inhibitor approved for treatment of HIV/AIDS patients who fail to respond to the current antiretroviral drugs. However, its clinical application is limited because of short half-life, drug resistance and cross-reactivity with the preexisting antibodies in HIV-infected patients. Using an artificial peptide strategy, we designed a peptide with non-native protein sequence, AP3, which exhibited potent antiviral activity against a broad spectrum of HIV-1 strains, including those resistant to T20, and had remarkably longer in vivo half-life than T20. While the preexisting antibodies in HIV-infected patients significantly suppressed T20's antiviral activity, these antibodies neither recognized AP3, nor attenuated its anti-HIV-1 activity. Structurally different from T20, AP3 could fold into single-helix and interact with gp41 NHR. The two residues, Met and Thr, at the N-terminus of AP3 form a hook-like structure to stabilize interaction between AP3 and NHR helices. Therefore, AP3 has potential for further development as a new HIV fusion inhibitor with improved antiviral efficacy, resistance profile and pharmacological properties over enfuvirtide. Meanwhile, this study highlighted the advantages of artificially designed peptides, and confirmed that this strategy could be used in developing artificial peptide-based viral fusion inhibitors against HIV and other enveloped viruses.
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