scholarly journals A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15

2018 ◽  
Author(s):  
P. De-la-Torre ◽  
D. Choudhary ◽  
R. Araya-Secchi ◽  
Y. Narui ◽  
M. Sotomayor
Keyword(s):  
Protein Interactions ◽  
Analytical Ultracentrifugation ◽  
Size Exclusion ◽  
Molecular Architecture ◽  
Protein Protein Interactions ◽  
Tip Link ◽  
Exclusion Chromatography ◽  
Protocadherin 15 ◽  
Cadherin 23 ◽  
Dynamics Simulations

ABSTRACTThe cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) repeats that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of non-classical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has eleven EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size exclusion chromatography coupled to multi-angle laser light scattering and small-angle X-ray scattering corroborate the crystallographic dimer and show that MAD12 induces parallel dimerization of PCDH15 near its membrane insertion point. In addition, steered molecular dynamics simulations suggest that MAD12 is mechanically weak and may unfold before tip-link rupture. Sequence analyses and structural modeling predict the existence of similar domains in cadherin-23, protocadherin-24, and the “giant” FAT and CELSR cadherins, indicating that some of them may also exhibit MAD-induced parallel dimerization.

scholarly journals Biochemical, Biophysical, and Mutational Analyses of Subunit Interactions of the Human Cytomegalovirus Nuclear Egress Complex

2009 ◽  
Vol 83 (7) ◽  
pp. 2996-3006 ◽  
Author(s):  
My D. Sam ◽  
Brady T. Evans ◽  
Donald M. Coen ◽  
James M. Hogle
Keyword(s):  
Human Cytomegalovirus ◽  
Protein Interactions ◽  
Analytical Ultracentrifugation ◽  
Size Exclusion ◽  
Titration Calorimetry ◽  
Protein Protein Interactions ◽  
Truncated Form ◽  
Nuclear Egress ◽  
Exclusion Chromatography

ABSTRACT Nuclear egress, the trafficking of herpesvirus nucleocapsids from the nucleus to the cytoplasm, involves two conserved viral proteins that form a complex at the nuclear envelope, referred to as the nuclear egress complex. In human cytomegalovirus, these two proteins are called UL50 and UL53. To study UL50 and UL53 in molecular detail, these proteins were expressed in bacteria and purified. To obtain highly expressed, pure proteins, it was necessary to truncate both constructs based on sequence conservation and predicted secondary structural elements. Size exclusion chromatography and analytical ultracentrifugation studies indicated that the truncated form of UL50 is a monomer in solution, that the truncated form of UL53 is a homodimer, and that, when mixed, the two proteins form a heterodimer. To identify residues of UL53 crucial for homodimerization and for heterodimerization with UL50, we constructed and expressed mutant forms of UL53 containing alanine substitutions in a predicted helix. Isothermal titration calorimetry was used to measure the binding affinities of the UL53 mutants to UL50. UL53 residues, the replacement of which reduced binding to UL50, form a surface on one face of the predicted helix. Moreover, most of the substitutions that reduce UL53-UL50 interactions also reduced homodimerization. Substitutions that reduced the interaction between UL50 and UL53 in vitro also reduced colocalization of full-length UL50 and UL53 at the nuclear rim in transfected cells. These results demonstrate direct protein-protein interactions between these proteins that are likely to be mediated by a helix, and they have implications for understanding nuclear egress and for drug discovery.

Oligomeric assembly and interactions within the human RuvB-like RuvBL1 and RuvBL2 complexes

2010 ◽  
Vol 429 (1) ◽  
pp. 113-125 ◽  
Author(s):  
Andrew Niewiarowski ◽  
Alison S. Bradley ◽  
Jayesh Gor ◽  
Adam R. McKay ◽  
Stephen J. Perkins ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Structural Model ◽  
Analytical Ultracentrifugation ◽  
Protein Complexes ◽  
Adenine Nucleotides ◽  
Size Exclusion ◽  
Molecular Architecture ◽  
Essential Components ◽  
Exclusion Chromatography

The two closely related eukaryotic AAA+ proteins (ATPases associated with various cellular activities), RuvBL1 (RuvB-like 1) and RuvBL2, are essential components of large multi-protein complexes involved in diverse cellular processes. Although the molecular mechanisms of RuvBL1 and RuvBL2 function remain unknown, oligomerization is likely to be important for their function together or individually, and different oligomeric forms might underpin different functions. Several experimental approaches were used to investigate the molecular architecture of the RuvBL1–RuvBL2 complex and the role of the ATPase-insert domain (domain II) for its assembly and stability. Analytical ultracentrifugation showed that RuvBL1 and RuvBL2 were mainly monomeric and each monomer co-existed with small proportions of dimers, trimers and hexamers. Adenine nucleotides induced hexamerization of RuvBL2, but not RuvBL1. In contrast, the RuvBL1–RuvBL2 complexes contained single- and double-hexamers together with smaller forms. The role of domain II in complex assembly was examined by size-exclusion chromatography using deletion mutants of RuvBL1 and RuvBL2. Significantly, catalytically competent dodecameric RuvBL1–RuvBL2, complexes lacking domain II in one or both proteins could be assembled but the loss of domain II in RuvBL1 destabilized the dodecamer. The composition of the RuvBL1–RuvBL2 complex was analysed by MS. Several species of mixed RuvBL1/2 hexamers with different stoichiometries were seen in the spectra of the RuvBL1–RuvBL2 complex. A number of our results indicate that the architecture of the human RuvBL1–RuvBL2 complex does not fit the recent structural model of the yeast Rvb1–Rvb2 complex.

scholarly journals Quantifying Protein-Protein Interactions in Molecular Simulations

2019 ◽  
Author(s):  
Alfredo Jost Lopez ◽  
Patrick K. Quoika ◽  
Max Linke ◽  
Gerhard Hummer ◽  
Juergen Koefinger
Keyword(s):  
Molecular Dynamics ◽  
Protein Interactions ◽  
Virial Coefficient ◽  
Analytical Ultracentrifugation ◽  
Molecular Simulations ◽  
Simple Expression ◽  
Protein Protein Interactions ◽  
Explicit Solvent ◽  
Dynamics Simulations ◽  
Crowded Environments

<p><a></a></p><p><a></a>We present simple, accurate, and efficient methods to estimate the dissociation constant K<sub>d </sub>and the second osmotic virial coefficient B<sub>2 </sub>from molecular simulations. We show that for simulations of two proteins in a box, K<sub>d </sub>is determined by B<sub>2 </sub>and the fraction of bound protein. We present two different methods to calculate B<sub>2 </sub>from Monte Carlo and molecular dynamics simulations using implicit or explicit solvent. We derive a surprisingly simple expression for B<sub>2</sub>, adding significantly to the understanding of this important quantity. Non-binding interactions of proteins and other macromolecules shape the physicochemical properties of the crowded environments inside cells and of biomolecular condensates. We show how to extract the contributions of non-binding conformations to B<sub>2 </sub>and discuss how these can be determined in analytical ultracentrifugation and SAXS experiments. We expect that our methods will prove to be instrumental in force parameterization efforts and high-throughput studies of large interactomes. </p>

scholarly journals Quantifying Protein-Protein Interactions in Molecular Simulations

2019 ◽  
Author(s):  
Alfredo Jost Lopez ◽  
Patrick K. Quoika ◽  
Max Linke ◽  
Gerhard Hummer ◽  
Juergen Koefinger
Keyword(s):  
Molecular Dynamics ◽  
Protein Interactions ◽  
Virial Coefficient ◽  
Analytical Ultracentrifugation ◽  
Molecular Simulations ◽  
Simple Expression ◽  
Protein Protein Interactions ◽  
Explicit Solvent ◽  
Dynamics Simulations ◽  
Crowded Environments

<p><a></a></p><p><a></a>We present simple, accurate, and efficient methods to estimate the dissociation constant K<sub>d </sub>and the second osmotic virial coefficient B<sub>2 </sub>from molecular simulations. We show that for simulations of two proteins in a box, K<sub>d </sub>is determined by B<sub>2 </sub>and the fraction of bound protein. We present two different methods to calculate B<sub>2 </sub>from Monte Carlo and molecular dynamics simulations using implicit or explicit solvent. We derive a surprisingly simple expression for B<sub>2</sub>, adding significantly to the understanding of this important quantity. Non-binding interactions of proteins and other macromolecules shape the physicochemical properties of the crowded environments inside cells and of biomolecular condensates. We show how to extract the contributions of non-binding conformations to B<sub>2 </sub>and discuss how these can be determined in analytical ultracentrifugation and SAXS experiments. We expect that our methods will prove to be instrumental in force parameterization efforts and high-throughput studies of large interactomes. </p>

scholarly journals Structural determinants of protocadherin-15 mechanics and function in hearing and balance perception

2020 ◽  
Vol 117 (40) ◽  
pp. 24837-24848
Author(s):  
Deepanshu Choudhary ◽  
Yoshie Narui ◽  
Brandon L. Neel ◽  
Lahiru N. Wimalasena ◽  
Carissa F. Klanseck ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Inner Ear ◽  
Sensory Perception ◽  
Head Movements ◽  
Structural Determinants ◽  
Tip Link ◽  
Protocadherin 15 ◽  
Cadherin 23 ◽  
Dynamics Simulations

The vertebrate inner ear, responsible for hearing and balance, is able to sense minute mechanical stimuli originating from an extraordinarily broad range of sound frequencies and intensities or from head movements. Integral to these processes is the tip-link protein complex, which conveys force to open the inner-ear transduction channels that mediate sensory perception. Protocadherin-15 and cadherin-23, two atypically large cadherins with 11 and 27 extracellular cadherin (EC) repeats, are involved in deafness and balance disorders and assemble as parallel homodimers that interact to form the tip link. Here we report the X-ray crystal structure of a protocadherin-15 + cadherin-23 heterotetrameric complex at 2.9-Å resolution, depicting a parallel homodimer of protocadherin-15 EC1-3 molecules forming an antiparallel complex with two cadherin-23 EC1-2 molecules. In addition, we report structures for 10 protocadherin-15 fragments used to build complete high-resolution models of the monomeric protocadherin-15 ectodomain. Molecular dynamics simulations and validated crystal contacts are used to propose models for the complete extracellular protocadherin-15 parallel homodimer and the tip-link bond. Steered molecular dynamics simulations of these models suggest conditions in which a structurally diverse and multimodal protocadherin-15 ectodomain can act as a stiff or soft gating spring. These results reveal the structural determinants of tip-link–mediated inner-ear sensory perception and elucidate protocadherin-15’s structural and adhesive properties relevant in disease.

scholarly journals Molecular dynamics shows complex interplay and long-range effects of post-translational modifications in yeast protein interactions

2021 ◽  
Vol 17 (5) ◽  
pp. e1008988
Author(s):  
Nikolina ŠoŠtarić ◽  
Vera van Noort
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Protein Structures ◽  
Cellular Protein ◽  
Free Energy Calculations ◽  
Protein Protein Interactions ◽  
Post Translational Modifications ◽  
Protein Properties ◽  
Dynamics Simulations

Post-translational modifications (PTMs) play a vital, yet often overlooked role in the living cells through modulation of protein properties, such as localization and affinity towards their interactors, thereby enabling quick adaptation to changing environmental conditions. We have previously benchmarked a computational framework for the prediction of PTMs’ effects on the stability of protein-protein interactions, which has molecular dynamics simulations followed by free energy calculations at its core. In the present work, we apply this framework to publicly available data on Saccharomyces cerevisiae protein structures and PTM sites, identified in both normal and stress conditions. We predict proteome-wide effects of acetylations and phosphorylations on protein-protein interactions and find that acetylations more frequently have locally stabilizing roles in protein interactions, while the opposite is true for phosphorylations. However, the overall impact of PTMs on protein-protein interactions is more complex than a simple sum of local changes caused by the introduction of PTMs and adds to our understanding of PTM cross-talk. We further use the obtained data to calculate the conformational changes brought about by PTMs. Finally, conservation of the analyzed PTM residues in orthologues shows that some predictions for yeast proteins will be mirrored to other organisms, including human. This work, therefore, contributes to our overall understanding of the modulation of the cellular protein interaction networks in yeast and beyond.

Molecular interactions between multihaem cytochromes: probing the protein–protein interactions between pentahaem cytochromes of a nitrite reductase complex

2011 ◽  
Vol 39 (1) ◽  
pp. 263-268 ◽  
Author(s):  
Colin Lockwood ◽  
Julea N. Butt ◽  
Thomas A. Clarke ◽  
David J. Richardson
Keyword(s):  
Protein Interactions ◽  
Nitrite Reductase ◽  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Protein Protein Interactions ◽  
Physiological Conditions ◽  
Sulfate Reducing ◽  
Redox Partner ◽  
Electron Reduction ◽  
Reducing Bacteria

The cytochrome c nitrite reductase NrfA is a 53 kDa pentahaem enzyme that crystallizes as a decahaem homodimer. NrfA catalyses the reduction of NO2− to NH4+ through a six electron reduction pathway that is of major physiological significance to the anaerobic metabolism of enteric and sulfate reducing bacteria. NrfA receives electrons from the 21 kDa pentahaem NrfB donor protein. This requires that redox complexes form between the NrfA and NrfB pentahaem cytochromes. The formation of these complexes can be monitored using a range of methodologies for studying protein–protein interactions, including dynamic light scattering, gel filtration, analytical ultracentrifugation and visible spectroscopy. These methods have been used to show that oxidized NrfA exists in dynamic monomer–dimer equilibrium with a Kd (dissociation constant) of 4 μM. Significantly, the monomeric and dimeric forms of NrfA are equally active for either the six electron reduction of NO2− or HSO3−. When mixed together, NrfA and NrfB exist in equilibrium with NrfAB, which is described by a Kd of 50 nM. Thus, since NrfA and NrfB are present in micromolar concentrations in the periplasmic compartment, it is likely that NrfB remains tightly associated with its NrfA redox partner under physiological conditions.

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