Chapter 3Coarse-Grained Modeling of Large Protein Complexes for Understanding Their Conformational Dynamics

Structural and biochemical studies have recently revealed a range of rationally engineered nanobodies with efficient neutralizing capacity against SARS-CoV-2 virus and resilience against mutational escape. In this work, we combined atomistic simulations and conformational dynamics analysis with the ensemble-based mutational profiling of binding interactions for a diverse panel of SARS-CoV-2 spike complexes with nanobodies. Using this computational toolkit, we identified dynamic signatures and binding affinity fingerprints for the SARS-CoV-2 spike protein complexes with nanobodies Nb6 and Nb20, VHH E, a pair combination VHH E+U, a biparatopic nanobody VHH VE, and a combination of CC12.3 antibody and VHH V/W nanobodies. Through ensemble-based deep mutational profiling of stability and binding affinities, we identify critical hotspots and characterize molecular mechanisms of SARS-CoV-2 spike protein binding with single ultra-potent nanobodies, nanobody cocktails and biparatopic nanobodies. By quantifying dynamic and energetic determinants of the SARS-CoV-2 S binding with nanobodies, we also examine the effects of circulating variants and escaping mutations. We found that mutational escape mechanisms may be controlled through structurally and energetically adaptable binding hotspots located in the host receptor-accessible binding epitope that are dynamically coupled to the stability centers in the distant epitope targeted by VHH U/V/W nanobodies. The results of this study suggested a mechanism in which through cooperative dynamic changes, nanobody combinations and biparatopic nanobody can modulate the global protein response and induce the increased resilience to common escape mutants.

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The right place at the right time: Aurora B kinase localization to centromeres and kinetochores

Essays in Biochemistry ◽

10.1042/ebc20190081 ◽

2020 ◽

Vol 64 (2) ◽

pp. 299-311 ◽

Cited By ~ 2

Author(s):

Amanda J. Broad ◽

Jennifer G. DeLuca

Keyword(s):

Spindle Assembly Checkpoint ◽

Protein Complexes ◽

Centromeric Heterochromatin ◽

Aurora B ◽

Mitotic Chromosomes ◽

Large Protein ◽

Aurora B Kinase ◽

Centromeric Protein ◽

Spindle Microtubules ◽

The Right

Abstract The fidelity of chromosome segregation during mitosis is intimately linked to the function of kinetochores, which are large protein complexes assembled at sites of centromeric heterochromatin on mitotic chromosomes. These key “orchestrators” of mitosis physically connect chromosomes to spindle microtubules and transduce forces through these connections to congress chromosomes and silence the spindle assembly checkpoint. Kinetochore-microtubule attachments are highly regulated to ensure that incorrect attachments are not prematurely stabilized, but instead released and corrected. The kinase activity of the centromeric protein Aurora B is required for kinetochore-microtubule destabilization during mitosis, but how the kinase acts on outer kinetochore substrates to selectively destabilize immature and erroneous attachments remains debated. Here, we review recent literature that sheds light on how Aurora B kinase is recruited to both centromeres and kinetochores and discuss possible mechanisms for how kinase interactions with substrates at distinct regions of mitotic chromosomes are regulated.

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Molecular requirements for the formation of a kinetochore–microtubule interface by Dam1 and Ndc80 complexes

The Journal of Cell Biology ◽

10.1083/jcb.201210091 ◽

2012 ◽

Vol 200 (1) ◽

pp. 21-30 ◽

Cited By ~ 49

Author(s):

Fabienne Lampert ◽

Christine Mieck ◽

Gregory M. Alushin ◽

Eva Nogales ◽

Stefan Westermann

Keyword(s):

Chromosome Segregation ◽

Protein Complexes ◽

Structural Elements ◽

Large Protein ◽

Kinetochore Assembly ◽

Sister Chromatids ◽

Dam1 Complex ◽

Shed Light ◽

Kinetochore Function

Kinetochores are large protein complexes that link sister chromatids to the spindle and transduce microtubule dynamics into chromosome movement. In budding yeast, the kinetochore–microtubule interface is formed by the plus end–associated Dam1 complex and the kinetochore-resident Ndc80 complex, but how they work in combination and whether a physical association between them is critical for chromosome segregation is poorly understood. Here, we define structural elements required for the Ndc80–Dam1 interaction and probe their function in vivo. A novel ndc80 allele, selectively impaired in Dam1 binding, displayed growth and chromosome segregation defects. Its combination with an N-terminal truncation resulted in lethality, demonstrating essential but partially redundant roles for the Ndc80 N-tail and Ndc80–Dam1 interface. In contrast, mutations in the calponin homology domain of Ndc80 abrogated kinetochore function and were not compensated by the presence of Dam1. Our experiments shed light on how microtubule couplers cooperate and impose important constraints on structural models for outer kinetochore assembly.

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Variable-Temperature Native Mass Spectrometry for Studies of Protein Folding, Stabilities, Assembly, and Molecular Interactions

Annual Review of Biophysics ◽

10.1146/annurev-biophys-102221-101121 ◽

2021 ◽

Vol 51 (1) ◽

Author(s):

Arthur Laganowsky ◽

David E. Clemmer ◽

David H. Russell

Keyword(s):

Mass Spectrometry ◽

Dynamic Range ◽

Protein Complexes ◽

Noncovalent Interactions ◽

Variable Temperature ◽

Regulatory Protein ◽

Conformational Dynamics ◽

Native Mass Spectrometry ◽

Annual Review ◽

Publication Date

The structures and conformational dynamics of proteins, protein complexes, and their noncovalent interactions with other molecules are controlled specifically by the Gibbs free energy (entropy and enthalpy) of the system. For some organisms, temperature is highly regulated, but the majority of biophysical studies are carried out at room, nonphysiological temperature. In this review, we describe variable-temperature electrospray ionization (vT-ESI) mass spectrometry (MS)-based studies with unparalleled sensitivity, dynamic range, and selectivity for studies of both cold- and heat-induced chemical processes. Such studies provide direct determinations of stabilities, reactivities, and thermodynamic measurements for native and non-native structures of proteins and protein complexes and for protein–ligand interactions. Highlighted in this review are vT-ESI-MS studies that reveal 40 different conformers of chymotrypsin inhibitor 2, a classic two-state (native → unfolded) unfolder, and thermochemistry for a model membrane protein system binding lipid and its regulatory protein. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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