scholarly journals Atomic resolution Protein Allostery from the multi-state Structure of a PDZ Domain

2021 ◽  
Author(s):  
Dzmitry Ashkinadze ◽  
Harindranath Kadavath ◽  
Celestine N Chi ◽  
Michael Friedmann ◽  
Dean Strotz ◽  
...  
Keyword(s):  
Nuclear Overhauser Effect ◽  
Tyrosine Phosphatase ◽  
Pdz Domain ◽  
Protein Structures ◽  
Atomic Resolution ◽  
Free Form ◽  
Protein Allostery ◽  
Correlated Motion ◽  
Partial Overlap ◽  
Α Helix

Recent methodological advances in solution NMR allow the determination of multi-state protein structures and provide insights into correlated motion at atomic resolution as demonstrated here for the well-studied PDZ2 domain of protein human tyrosine phosphatase 1E for which protein allostery was predicted. Two-state protein structures were calculated for both the free form and in complex with the RA-GEF2 peptide using the exact nuclear Overhauser effect (eNOE) method. In the apo protein states an allosteric conformational preselection step comprising almost 60% of the domain was detected with an "open" ligand welcoming state and a "closed" state that obstructs the binding site by the distance between the β-sheet, α-helix 2 and sidechains of residues Lys38 and Lys72. Observed apo-holo structural rearrangements of induced fit-type are in line with previously published evolution-based analysis covering ~25% of the domain with only a partial overlap with the protein allostery of the open form. These presented structural studies highlight the presence of a dedicated highly optimized dynamic interplay of the complexity of the PDZ2 domain owed by the structure-dynamics landscape.

scholarly journals MicroED Structures from Micrometer Thick Protein Crystals

2017 ◽  
Author(s):  
Michael W. Martynowycz ◽  
Calina Glynn ◽  
Jennifer Miao ◽  
M. Jason de la Cruz ◽  
Johan Hattne ◽  
...  
Keyword(s):  
Theoretical Calculations ◽  
Protein Structures ◽  
Atomic Resolution ◽  
Proteinase K ◽  
Protein Crystals ◽  
Molecular Replacement ◽  
Varying Thickness ◽  
Order Of Magnitude ◽  
Human Prion Protein ◽  
Do So

AbstractTheoretical calculations suggest that crystals exceeding 100 nm thickness are excluded by dynamical scattering from successful structure determination using microcrystal electron diffraction (MicroED). These calculations are at odds with experimental results where MicroED structures have been determined from significantly thicker crystals. Here we systematically evaluate the influence of thickness on the accuracy of MicroED intensities and the ability to determine structures from protein crystals one micrometer thick. To do so, we compare ab initio structures of a human prion protein segment determined from thin crystals to those determined from crystals up to one micrometer thick. We also compare molecular replacement solutions from crystals of varying thickness for a larger globular protein, proteinase K. Our results indicate that structures can be reliably determined from crystals at least an order of magnitude thicker than previously suggested by simulation, opening the possibility for an even broader range of MicroED experiments.SummaryAtomic resolution protein structures can be determined by MicroED from crystals that surpass the theoretical maximum thickness limit by an order of magnitude.

scholarly journals Protein Structure Determination in Living Cells

2019 ◽  
Vol 20 (10) ◽  
pp. 2442 ◽  
Author(s):  
Teppei Ikeya ◽  
Peter Güntert ◽  
Yutaka Ito
Keyword(s):  
Protein Structure ◽  
Structure Determination ◽  
Structure Prediction ◽  
Structural Information ◽  
Nuclear Overhauser Effect ◽  
Protein Structures ◽  
Three Dimensional ◽  
Structural Data ◽  
Sample Tube ◽  
In Cells

To date, in-cell NMR has elucidated various aspects of protein behaviour by associating structures in physiological conditions. Meanwhile, current studies of this method mostly have deduced protein states in cells exclusively based on ‘indirect’ structural information from peak patterns and chemical shift changes but not ‘direct’ data explicitly including interatomic distances and angles. To fully understand the functions and physical properties of proteins inside cells, it is indispensable to obtain explicit structural data or determine three-dimensional (3D) structures of proteins in cells. Whilst the short lifetime of cells in a sample tube, low sample concentrations, and massive background signals make it difficult to observe NMR signals from proteins inside cells, several methodological advances help to overcome the problems. Paramagnetic effects have an outstanding potential for in-cell structural analysis. The combination of a limited amount of experimental in-cell data with software for ab initio protein structure prediction opens an avenue to visualise 3D protein structures inside cells. Conventional nuclear Overhauser effect spectroscopy (NOESY)-based structure determination is advantageous to elucidate the conformations of side-chain atoms of proteins as well as global structures. In this article, we review current progress for the structure analysis of proteins in living systems and discuss the feasibility of its future works.

Conformational changes in the third PDZ domain of the neuronal postsynaptic density protein 95

2019 ◽  
Vol 75 (4) ◽  
pp. 381-391 ◽  
Author(s):  
Ana Camara-Artigas ◽  
Javier Murciano-Calles ◽  
Jose C. Martínez
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Pdz Domain ◽  
Postsynaptic Density ◽  
Crystal Form ◽  
Space Groups ◽  
The Third ◽  
Conformational Plasticity ◽  
Α Helix ◽  
Postsynaptic Density Protein

PDZ domains are protein–protein recognition modules that interact with other proteins through short sequences at the carboxyl terminus. These domains are structurally characterized by a conserved fold composed of six β-strands and two α-helices. The third PDZ domain of the neuronal postsynaptic density protein 95 has an additional α-helix (α3), the role of which is not well known. In previous structures, a succinimide was identified in the β2–β3 loop instead of Asp332. The presence of this modified residue results in conformational changes in α3. In this work, crystallographic structures of the following have been solved: a truncated form of the third PDZ domain of the neuronal postsynaptic density protein 95 from which α3 has been removed, D332P and D332G variants of the protein, and a new crystal form of this domain showing the binding of Asp332 to the carboxylate-binding site of a symmetry-related molecule. Crystals of the wild type and variants were obtained in different space groups, which reflects the conformational plasticity of the domain. Indeed, the overall analysis of these structures suggests that the conformation of the β2–β3 loop is correlated with the fold acquired by α3. The alternate conformation of the β2–β3 loop affects the electrostatics of the carboxylate-binding site and might modulate the binding of different PDZ-binding motifs.

Protein phasing at non-atomic resolution by combining Patterson andVLDtechniques

2014 ◽  
Vol 70 (7) ◽  
pp. 1994-2006 ◽  
Author(s):  
Rocco Caliandro ◽  
Benedetta Carrozzini ◽  
Giovanni Luca Cascarano ◽  
Giuliana Comunale ◽  
Carmelo Giacovazzo ◽  
...  
Keyword(s):  
Model Building ◽  
Protein Structures ◽  
Experimental Information ◽  
Atomic Resolution ◽  
Data Sets ◽  
Density Maps ◽  
Combined Use ◽  
Final Electron ◽  
Automatic Model Building

Phasing proteins at non-atomic resolution is still a challenge for anyab initiomethod. A variety of algorithms [Patterson deconvolution, superposition techniques, a cross-correlation function (Cmap), theVLD(vive la difference) approach, the FF function, a nonlinear iterative peak-clipping algorithm (SNIP) for defining the background of a map and thefree lunchextrapolation method] have been combined to overcome the lack of experimental information at non-atomic resolution. The method has been applied to a large number of protein diffraction data sets with resolutions varying from atomic to 2.1 Å, with the condition that S or heavier atoms are present in the protein structure. The applications include the use ofARP/wARPto check the quality of the final electron-density maps in an objective way. The results show that resolution is still the maximum obstacle to protein phasing, but also suggest that the solution of protein structures at 2.1 Å resolution is a feasible, even if still an exceptional, task for the combined set of algorithms implemented in the phasing program. The approach described here is more efficient than the previously described procedures:e.g.the combined use of the algorithms mentioned above is frequently able to provide phases of sufficiently high quality to allow automatic model building. The method is implemented in the current version ofSIR2014.

Protein Allostery at Atomic Resolution

2020 ◽  
Vol 132 (49) ◽  
pp. 22316-22323
Author(s):  
Dean Strotz ◽  
Julien Orts ◽  
Harindranath Kadavath ◽  
Michael Friedmann ◽  
Dhiman Ghosh ◽  
...  
Keyword(s):  
Atomic Resolution ◽  
Protein Allostery

Structure of a double-domain phosphagen kinase reveals an asymmetric arrangement of the tandem domains

2015 ◽  
Vol 71 (4) ◽  
pp. 779-789 ◽  
Author(s):  
Zhiming Wang ◽  
Zhu Qiao ◽  
Sheng Ye ◽  
Rongguang Zhang
Keyword(s):  
Bound States ◽  
Hydrophobic Interactions ◽  
Crystal Packing ◽  
Protein Structures ◽  
Structural Basis ◽  
Phosphagen Kinase ◽  
Domain Arrangement ◽  
Α Helix ◽  
Dynamics Simulations ◽  
Shaped Domain

Tandem duplications and fusions of single genes have led to magnificent expansions in the divergence of protein structures and functions over evolutionary timescales. One of the possible results is polydomain enzymes with interdomain cooperativities, few examples of which have been structurally characterized at the full-length level to explore their innate synergistic mechanisms. This work reports the crystal structures of a double-domain phosphagen kinase in both apo and ligand-bound states, revealing a novel asymmetric L-shaped arrangement of the two domains. Unexpectedly, the interdomain connections are not based on a flexible hinge linker but on a rigid secondary-structure element: a long α-helix that tethers the tandem domains in relatively fixed positions. Besides the connective helix, the two domains also contact each other directly and form an interdomain interface in which hydrogen bonds and hydrophobic interactions further stabilize the L-shaped domain arrangement. Molecular-dynamics simulations show that the interface is generally stable, suggesting that the asymmetric domain arrangement crystallographically observed in the present study is not a conformational state simply restrained by crystal-packing forces. It is possible that the asymmetrically arranged tandem domains could provide a structural basis for further studies of the interdomain synergy.

scholarly journals Degradation of Tyrosine Phosphatase PTPN3 (PTPH1) by Association with Oncogenic HumanPapillomavirus E6 Proteins

2006 ◽  
Vol 81 (5) ◽  
pp. 2231-2239 ◽  
Author(s):  
Ming Jing ◽  
Joanna Bohl ◽  
Nicole Brimer ◽  
Michael Kinter ◽  
Scott B. Vande Pol
Keyword(s):  
Growth Factor ◽  
Ubiquitin Ligase ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Pdz Domain ◽  
Hect Domain ◽  
Hpv E6 ◽  
Colon Cancers ◽  
Tumor Viruses

ABSTRACT Oncoproteins from DNA tumor viruses associate with critical cellular proteins to regulate cell proliferation, survival, and differentiation.Human papillomavirus (HPV) E6 oncoproteins have been previously shown to associate with a cellular HECT domain ubiquitin ligase termed E6AP (UBE3A). Here we show that the E6-E6AP complex associates with and targets the degradation of the protein tyrosine phosphatase PTPN3 (PTPH1) in vitro and in living cells. PTPN3 is a membrane-associated tyrosine phosphatase with FERM, PDZ, and PTP domains previously implicated in regulating tyrosine phosphorylation of growth factor receptors and p97 VCP (valosin-containing protein, termed Cdc48 in Saccharomyces cerevisiae) and is mutated in a subset of colon cancers. Degradation of PTPN3 by E6 requires E6AP, the proteasome, and an interaction between the carboxy terminus of E6 and the PDZ domain of PTPN3. In transduced keratinocytes, E6 confers reduced growth factor requirements, a function that requires the PDZ ligand of E6 and that can in part be replicated by inhibiting the expression of PTPN3. This report demonstrates the potential of E6 to regulate phosphotyrosine metabolism through the targeted degradation of a tyrosine phosphatase.

scholarly journals Constructing synthetic-protein assemblies from de novo designed 310 helices

2021 ◽  
Author(s):  
Prasun Kumar ◽  
Neil G. Paterson ◽  
Jonathan Clayden ◽  
Derek N. Woolfson
Keyword(s):  
Rational Design ◽  
De Novo ◽  
Protein Structures ◽  
Water Soluble ◽  
Design Solution ◽  
Protein Assemblies ◽  
Synthetic Protein ◽  
Hydrophobic Cores ◽  
Α Helix ◽  
Helical Folding

Compared with the iconic α helix, 310 helices occur much less frequently in protein structures. The different 310-helical parameters lead to energetically less favourable internal energies, and a reduced tendency to pack into defined higher-order structures. Consequently, in natural proteins, 310 helices rarely extend past 6 residues, and do not form regular supersecondary, tertiary, or quaternary interactions. Here, we show that despite their absence in nature, synthetic protein-like assemblies can be built from 310 helices. We report the rational design, solution-phase characterisation, and an X-ray crystal structure for water-soluble bundles of 310 helices with consolidated hydrophobic cores. The design uses 6-residue repeats informed by analysing natural 310 helices, and incorporates aminoisobutyric acid residues. Design iterations reveal a tipping point between α-helical and 310-helical folding, and identify features required for stabilising assemblies in this unexplored region of protein-structure space.

scholarly journals Crystal structure of Rv3899c184–410, a hypothetical protein fromMycobacterium tuberculosis

2016 ◽  
Vol 72 (8) ◽  
pp. 642-645 ◽  
Author(s):  
Yingying Liu ◽  
Yunrong Gao ◽  
Defeng Li ◽  
Joy Fleming ◽  
Honglin Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
Biological Function ◽  
Vaccine Candidate ◽  
Protein Structures ◽  
Hypothetical Protein ◽  
Helix Bundle ◽  
Potential Vaccine ◽  
Α Helix ◽  
New Protein

Rv3899c is a hypothetical protein fromMycobacterium tuberculosiswhich is conserved across mycobacteria. It is predicted to be secreted and has been found in culture filtrates. It has been proposed as a potential vaccine candidate; however, its biological function is unknown. Here, the global structure of Rv3899c184–410, a fragment of Rv3899c, is reported. The structure resembles the shell of a sea snail, and its N- and C-termini form two relatively independent compact domains: an α/β/α sandwich folding domain and an α-helix bundle domain. There are no reported protein structures for any Rv3899c homologues; this structure provides the first structural glimpse of a new protein family consisting of Rv3899c and its homologues.

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