key: cord-0074760-navycaze
authors: Kim, Seonghan; Liu, Yi; Lei, Zewei; Dicker, Jeffrey; Cao, Yiwei; Zhang, Xiaohui; Im, Wonpil
title: Differential interactions between human ACE2 and spike RBD of SARS-CoV-2 variants of concern
date: 2022-02-11
journal: Biophys J
DOI: 10.1016/j.bpj.2021.11.2515
sha: be45659984f768dfcfb2b8db35493f8c4ff9e2d2
doc_id: 74760
cord_uid: navycaze

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identify changes in interfaces associated with linker substitutions and surface mutations while single-molecule FRET probes the exchange rates between conformational states. The effects linker mutations within the PDZ12 tandem provide insights into linkers' energetics and functional implications in modulating supertertiary structure and potential regulatory functions in complex intermolecular interactions.

A structure-function approach to elucidate the molecular mechanism of the telomere C-strand fill-in process Qixiang He 1 , Andrey Baranovskiy 2 , Xiuhua Lin 1 , Ben Lusk 1 , Victoria Tholkes 1 , Tahir Tahirov 2 , Ci Ji Lim 1 . 1 Department of Biochemistry, University of Wisconsin Madison, Madison, WI, USA, 2 Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA. Telomeres are repetitive DNA sequences decorated with telomere-specific proteins at the ends of linear chromosomes in eukaryotic cells. The regulation of human telomere length is essential for maintaining genome stability and cellular lifespan, and its dysregulation causes premature aging and cancer. The heterotrimeric CTC1-STN1-TEN1 (CST) protein complex is vital for the timely termination of telomere elongation by telomerase. CST is also required to recruit DNA polymerase a-primase (pol a-primase) to the newly synthesized telomeric G-overhang ssDNA to convert the telomeric ssDNA to dsDNA (known as C-strand fill-in). Due to the lack of biochemical and structural knowledge on CST-pol a-primase, it is unknown how the complementary Cstrand at telomeric G-overhang is synthesized by CST-pol a-primase machinery. To answer this question, we combine biochemical and cryo-electron microscopy (cryo-EM) methods to investigate the molecular mechanisms of human Pol a-primase recruitment to telomeres and its activity stimulation by CST. We show recombinant CST directly interacts with Pol a-primase, and a single-stranded telomeric DNA is not needed to form the co-complex. We also used cryo-EM single-particle analysis to solve a catalytic state of Pol a-primase engaged to a DNA/RNA molecule.

Differential interactions between human ACE2 and spike RBD of SARS-CoV-2 variants of concern Seonghan Kim 1 , Yi Liu 1 , Zewei Lei 1 , Jeffrey Dicker 1 , Yiwei Cao 2 , Xiaohui Zhang 1 , Wonpil Im 3 . Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. It is known that the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 interacts with the human angiotensin-converting enzyme 2 (ACE2) receptor, initiating the entry of SARS-CoV-2. Since its emergence, a variety of SARS-CoV-2 variants have been identified, and the variants showing high infectivity and possessing potential diagnostic impact are classified as the variants of concern (VOC) and the variants of interest (VOI) by the US CDC. This work characterizes distinctive binding interactions between ACE2 and RBD of all current VOC (Alpha, Beta, Gamma, and Delta) and two VOI (Epsilon and Kappa) employing both all-atom steered molecular dynamics (SMD) simulations and microscale thermophoresis (MST) experiments. We report that the RBD of the Alpha (N501Y) variant requires the highest amount of forces to be detached from ACE2 due to the N501Y mutation in addition to the role of N90-glycan, followed by Beta/Gamma (K417N/T, E484K, and N501Y) or Delta (L452R and T478K) variant. The RBD of the Epsilon (L452R) variant is relatively easily detached from ACE2 compared to other variants. Our SMD simulations and MST experiments reveal what makes each variant more contagious in terms of RBD and ACE2 interactions. This study provides valuable information that distinguishes important features of all variants in terms of RBD-ACE2 interactions and sheds a light on developing new drugs to inhibit SARS-CoV-2 entry effectively.

Higher-order structure analysis of high concentration monoclonal antibody by circular dichroism (CD) and infrared (IR) spectroscopy Ai Yamane 1 , Taiji Oyama 1 , Kohei Tamura 1 , Miyuki Kanno 1 , Shingo Norimoto 1 , Forrest Kohl 2 , Satoko Suzuki 1 , Kenichi Akao 1 . 1 JASCO Corporation, Hachioji, Japan, 2 JASCO Inc., Easton, MD, USA. Antibody therapeutics have been dramatically expanding their market over the past decade and become one of the major biotherapeutics proteins. Circular dichroism (CD) spectroscopy is an easy and robust method to analyze the higher-order structure (HOS) of antibodies. It is used to check the HOS comparability/ homogeneity before and after a change in the manufacturing process as specified in ICH Q5E, and to compare the HOS between innovators and biosimilars as described in FDA, EMA and other guidelines. In addition, CD spectroscopy is a well-known technique to estimate the protein secondary structure. While CD measurement of protein is often performed with relatively low concentration up to about 1 to 5 mg/mL, most of the antibody therapeutics are formulated and prescribed with concentrations of 10 mg/mL or higher. In the case of prefilled syringe-type dosage form, which has become increasingly popular in recent years, the concentration can be above 100 mg/mL. It is known that proteins behave differently at high concentrations in comparison to dilute conditions, and there is a need to evaluate the HOS of antibody therapeutics at their prescribed concentration. In this study, we developed a method to study IgG with high concentration using far-UV and near-UV CD spectroscopy. A combination of the latest CD spectrometer J-1500 and the BeStSel algorithm [Beta Structure Selection, provided by Department of Biochemistry, Institute of Biology, Eötvös Loránd University] enabled the analysis of the secondary structure of antibodies at concentrations of 10 mg/ml or higher with high accuracy. The results of the secondary structure analysis were consistent with the ones obtained by FT-IR. Furthermore, we surprisingly succeeded in measuring near-UV CD spectrum of above 100 mg/ mL IgG to obtain the tertiary structure information.

Determinants of E3 ligase adaptor SPOP binding to intrinsically disordered substrates Emery T. Usher 1 , Scott A. Showalter 2 . 1 Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA, 2 Department of Chemistry, Pennsylvania State University, University Park, PA, USA. The E3 ubiquitin ligase adaptor SPOP plays a key role in the regulation of numerous transcription factors, cell cycle regulators, and other proteins. SPOP binds to target substrate proteins through its substrate-recognition MATH domain, and recruits ubiquitin ligases by its tandem dimerization domains. In particular, SPOP favors binding within intrinsically disordered regions (IDRs) of substrates; such IDRs often contain multiple SPOP-binding motifs, which enables high-affinity binding and processive ubiquitination activity through multivalent interactions. Through the study of a specific SPOP substrate, the pancreatic transcription factor Pdx1, our lab found SPOP may bind substrates with less specificity than previously understood. Additionally, we showed that certain intermolecular contacts outside of the substrate-binding cleft contribute nontrivially to SPOP-substrate stability. Therefore, we hypothesized that there are yet unstudied atomistic interactions between SPOP and substrates that contribute to the general and substrate-specific features of SPOP binding. To this end, I will characterize naturally-occurring cancer mutants of SPOP that fail to appropriately regulate their substrate proteins. I will use fluorescence anisotropy to evaluate changes in binding affinity linked to SPOP or substrate mutation. Urea denaturation experiments will assess the effect of SPOP point mutations on protein stability. Finally, I will employ simulations and experiments to probe the dynamics of wild-type SPOP in the bound and unbound forms. In all, this study should re-establish the molecular determinants of SPOP-substrate binding and simultaneously inform the molecular etiologies of SPOP-linked diseases.

Cyanobacteria generate circadian rhythms via a molecular clock that keeps track of time post-translationally. At the center of this clock is the hexameric AAAþ protein KaiC, which undergoes a cycle of autophosphorylation in its C-terminal domain (CII) that governs binding of the other clock proteins, KaiA and KaiB, at its N-terminal domain (CI) over the course of the 24-hour day. KaiA promotes autophosphorylation by interacting with C-terminal loops of KaiC, known as A-loops, until KaiA is sequestered by KaiB