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1. Structure analysis suggests Ess1 isomerizes the carboxy-terminal domain of RNA polymerase II via a bivalent anchoring mechanism

   https://doi.org/10.1038/s42003-021-01906-8  

   Namitz, Kevin E. ; Zheng, Tongyin ; Canning, Ashley J. ; Alicea-Velazquez, Nilda L. ; Castañeda, Carlos A. ; Cosgrove, Michael S. ; Hanes, Steven D. ( December 2021 , Communications Biology)

   null (Ed.)

   Abstract Accurate gene transcription in eukaryotes depends on isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II. Isomerization is part of the “CTD code” that regulates recruitment of proteins required for transcription and co-transcriptional RNA processing. Saccharomyces cerevisiae Ess1 and its human ortholog, Pin1, are prolyl isomerases that engage the long heptad repeat (YSPTSPS) 26 of the CTD by an unknown mechanism. Here, we used an integrative structural approach to decipher Ess1 interactions with the CTD. Ess1 has a rigid linker between its WW and catalytic domains that enforces a distance constraint for bivalent interaction with the ends of long CTD substrates (≥4–5 heptad repeats). Our binding results suggest that the Ess1 WW domain anchors the proximal end of the CTD substrate during isomerization, and that linker divergence may underlie evolution of substrate specificity. 

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2. Tuning Protein Dynamics to Sense Rapid Endoplasmic‐Reticulum Calcium Dynamics

   https://doi.org/10.1002/ange.202108443  

   Deng, Xiaonan ; Yao, Xin‐Qiu ; Berglund, Ken ; Dong, Bin ; Ouedraogo, Daniel ; Ghane, Mohammad A. ; Zhuo, You ; McBean, Cheyenne ; Wei, Zheng Zachory ; Gozem, Samer ; et al ( September 2021 , Angewandte Chemie)

   Multi‐scale calcium (Ca²⁺) dynamics, exhibiting wide‐ranging temporal kinetics, constitutes a ubiquitous mode of signal transduction. We report a novel endoplasmic‐reticulum (ER)‐targeted Ca²⁺indicator, R‐CatchER, which showed superior kinetics in vitro (k_off≥2×10³ s⁻¹,k_on≥7×10⁶ M⁻¹ s⁻¹) and in multiple cell types. R‐CatchER captured spatiotemporal ER Ca²⁺dynamics in neurons and hotspots at dendritic branchpoints, enabled the first report of ER Ca²⁺oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca²⁺‐based functional cooperativity of CaSR. We elucidate the mechanism of R‐CatchER and propose a principle to rationally design genetically encoded Ca²⁺indicators with a single Ca²⁺‐binding site and fast kinetics by tuning rapid fluorescent‐protein dynamics and the electrostatic potential around the chromophore. The design principle is supported by the development of G‐CatchER2, an upgrade of our previous (G‐)CatchER with improved dynamic range. Our work may facilitate protein design, visualizing Ca²⁺dynamics, and drug discovery.

    

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3. Tuning Protein Dynamics to Sense Rapid Endoplasmic‐Reticulum Calcium Dynamics

   https://doi.org/10.1002/anie.202108443  

   Deng, Xiaonan ; Yao, Xin‐Qiu ; Berglund, Ken ; Dong, Bin ; Ouedraogo, Daniel ; Ghane, Mohammad A. ; Zhuo, You ; McBean, Cheyenne ; Wei, Zheng Zachory ; Gozem, Samer ; et al ( September 2021 , Angewandte Chemie International Edition)

   Multi‐scale calcium (Ca²⁺) dynamics, exhibiting wide‐ranging temporal kinetics, constitutes a ubiquitous mode of signal transduction. We report a novel endoplasmic‐reticulum (ER)‐targeted Ca²⁺indicator, R‐CatchER, which showed superior kinetics in vitro (k_off≥2×10³ s⁻¹,k_on≥7×10⁶ M⁻¹ s⁻¹) and in multiple cell types. R‐CatchER captured spatiotemporal ER Ca²⁺dynamics in neurons and hotspots at dendritic branchpoints, enabled the first report of ER Ca²⁺oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca²⁺‐based functional cooperativity of CaSR. We elucidate the mechanism of R‐CatchER and propose a principle to rationally design genetically encoded Ca²⁺indicators with a single Ca²⁺‐binding site and fast kinetics by tuning rapid fluorescent‐protein dynamics and the electrostatic potential around the chromophore. The design principle is supported by the development of G‐CatchER2, an upgrade of our previous (G‐)CatchER with improved dynamic range. Our work may facilitate protein design, visualizing Ca²⁺dynamics, and drug discovery.

    

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4. Binding Site Maturation Modulated by Molecular Density Underlies Ndc80 Binding to Kinetochore Receptor CENP-T

   https://doi.org/10.1101/2024.02.25.581584  

   Tarasovetc, Ekaterina V ; Sissoko, Gunter B ; Maiorov, Aleksandr ; Mukhina, Anna S ; Ataullakhanov, Fazoil I ; Cheeseman, Iain M ; Grishchuk, Ekaterina L ( February 2024 , bioRxiv)

   Macromolecular assembly depends on tightly regulated pairwise binding interactions that are selectively favored at assembly sites while being disfavored in the soluble phase. This selective control can arise due to molecular density-enhanced binding, as recently found for the kinetochore scaffold protein CENP-T. When clustered, CENP-T recruits markedly more Ndc80 complexes than its monomeric counterpart, but the underlying molecular basis remains elusive. Here, we use quantitativein vitroassays to reveal two distinct mechanisms driving this behavior. First, Ndc80 binding to CENP-T is a two-step process: initially, Ndc80 molecules rapidly associate and dissociate from disordered N-terminal binding sites on CENP-T. Over time, these sites undergo maturation, resulting in stronger Ndc80 retention. Second, we find that this maturation transition is regulated by a kinetic barrier that is sensitive to the molecular environment. In the soluble phase, binding site maturation is slow, but within CENP-T clusters, this process is markedly accelerated. Notably, the two Ndc80 binding sites in human CENP-T exhibit distinct maturation rates and environmental sensitivities, which correlate with their different amino-acid content and predicted binding conformations. This clustering-induced maturation is evident in dividing human cells, suggesting a distinct regulatory entry point for controlling kinetochore assembly. We propose that the tunable acceleration of binding site maturation by molecular crowding may represent a general mechanism for promoting the formation of macromolecular structures.

   A distinctive mechanism of protein-protein interaction underpins the assembly of kinetochores, which is critical for human cell division. During mitosis, the Ndc80 complex must bind tightly to the unstructured N-terminus of its receptor, CENP-T, which is densely clustered at kinetochores. Using single-moleculein vitroassays, we show that Ndc80 binding is mediated by an initially unstable yet tunable interface. The high molecular density of CENP-T at the kinetochores accelerates the maturation of this binding interface, favoring the formation of stable complexes within the kinetochore structure, rather than in the soluble phase. This environment-driven modulation of binding site maturation may represent a key regulatory mechanism for ensuring strong and specific interactions during the assembly of macromolecular complexes such as kinetochores.

    

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5. Structural domains of SARS-CoV-2 nucleocapsid protein coordinate to compact long nucleic acid substrates

   https://doi.org/10.1093/nar/gkac1179  

   Morse, Michael ; Sefcikova, Jana ; Rouzina, Ioulia ; Beuning, Penny J. ; Williams, Mark C. ( December 2022 , Nucleic Acids Research)

   The SARS-CoV-2 nucleocapsid (N) protein performs several functions including binding, compacting, and packaging the ∼30 kb viral genome into the viral particle. N protein consists of two ordered domains, with the N terminal domain (NTD) primarily associated with RNA binding and the C terminal domain (CTD) primarily associated with dimerization/oligomerization, and three intrinsically disordered regions, an N-arm, a C-tail, and a linker that connects the NTD and CTD. We utilize an optical tweezers system to isolate a long single-stranded nucleic acid substrate to measure directly the binding and packaging function of N protein at a single molecule level in real time. We find that N protein binds the nucleic acid substrate with high affinity before oligomerizing and forming a highly compact structure. By comparing the activities of truncated protein variants missing the NTD, CTD, and/or linker, we attribute specific steps in this process to the structural domains of N protein, with the NTD driving initial binding to the substrate and ensuring high localized protein density that triggers interprotein interactions mediated by the CTD, which forms a compact and stable protein-nucleic acid complex suitable for packaging into the virion.

    

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