Comprehending the Structure, Dynamics, and Mechanism of Action of Drug-Resistant HIV Protease.

Since the emergence of the Human Immunodeficiency Virus (HIV) in the 1980s, strategies to combat HIV-AIDS are continuously evolving. Among the many tested targets to tackle this virus, its protease enzyme (PR) was proven to be an attractive option that brought about numerous research publications and ten FDA-approved drugs to inhibit the PR activity. However, the drug-induced mutations in the enzyme made these small molecule inhibitors ineffective with prolonged usage. The research on HIV PR, therefore, remains a thrust area even today. Through this review, we reiterate the importance of understanding the various structural and functional components of HIV PR in redesigning the structure-based small molecule inhibitors. We also discuss at length the currently available FDA-approved drugs and how these drug molecules induced mutations in the enzyme structure. We then recapitulate the reported mechanisms on how these drug-resistant variants remain sufficiently active to cleave the natural substrates. We end with the future scope covering the recently proposed strategies that show promise to deal with the mutations.

Structural basis for distinct operational modes and protease activation in AAA+ protease Lon.

Substrate-bound structures of AAA+ protein translocases reveal a conserved asymmetric spiral staircase architecture wherein a sequential ATP hydrolysis cycle drives hand-over-hand substrate translocation. However, this configuration is unlikely to represent the full conformational landscape of these enzymes, as biochemical studies suggest distinct conformational states depending on the presence or absence of substrate. Here, we used cryo-electron microscopy to determine structures of the Yersinia pestis Lon AAA+ protease in the absence and presence of substrate, uncovering the mechanistic basis for two distinct operational modes. In the absence of substrate, Lon adopts a left-handed, "open" spiral organization with autoinhibited proteolytic active sites. Upon the addition of substrate, Lon undergoes a reorganization to assemble an enzymatically active, right-handed "closed" conformer with active protease sites. These findings define the mechanistic principles underlying the operational plasticity required for processing diverse protein substrates.

Aurora A site specific TACC3 phosphorylation regulates astral microtubule assembly by stabilizing γ-tubulin ring complex.

BACKGROUND: Astral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. Previous studies have demonstrated that human transforming acidic coiled-coil 3 (TACC3), the most conserved TACC family protein, regulates formation of astral microtubules at centrosomes in vertebrate cells by affecting γ-tubulin ring complex (γ-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood. RESULTS: Here, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing γ-TuRC assembly in human cells. Mutation of the most conserved Aurora A targeting site, Ser 558 to alanine (S558A) in TACC3 results in robust loss of astral microtubules and disrupts localization of the γ-tubulin ring complex (γ-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the γ-TuRC proteins in a manner similar to control cells expressed with wild type TACC3. Time-lapse imaging reveals that S558A mutation leads to defects in positioning of the spindle-poles and thereby causes delay in metaphase to anaphase transition. Biochemical results determine that the Ser 558- phosphorylated TACC3 interacts with the γ-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of γ-TuRC from the small complex components. CONCLUSIONS: The results demonstrate that TACC3 phosphorylation stabilizes γ- tubulin ring complex assembly and thereby regulates formation of centrosomal asters. They also implicate a potential role of TACC3 phosphorylation in the functional integrity of centrosomes/spindle poles.