scholarly journals Development of an integrated structural biology platform specialized for sub-100 kDa protein complexes to support biologics discovery and rational engineering

2021 ◽  
Author(s):  
Yuri Iozzo ◽  
Egor Svidritskiy ◽  
Yu Qiu ◽  
Albert Xu ◽  
Anna Park ◽  
...  
Keyword(s):  
Decision Making ◽  
Structural Biology ◽  
Structural Information ◽  
Protein Complexes ◽  
Rapid Development ◽  
Protein Structures ◽  
Cost Effective ◽  
Particle Orientation ◽  
Dynamics Simulation ◽  
Rational Engineering

Abstract Background Developing a biologic medicine requires successful decision making during selection and optimization in addition to the pool of candidates at early research stages. Knowing structural information and binding patterns between drug target and discovery candidates greatly increases the probability of success. Methods With the cryo-EM resolution revolution and rapid development of computational software, we have evaluated and integrated various tools in structural biology and the computation field and established a highly cost-effective platform which allows us to obtain fast and accurate structural information for nearly all our biologics projects with a close to 100% success rate and as fast as weeks turn-around time. Results Here we report four case studies selected from 38 different protein structures and share how we integrate cryo-EM structure determination, computational structure modeling, and molecular dynamics simulation. With proper decision making and strategic planning, the platform allows us to obtain quality results within days to weeks, including sub-100 kDa complexes which are usually considered a challenge due to their small size. Conclusions Our utilization of this differential approach and multiple software packages allows us to manage priorities and resources to achieve goals quickly and efficiently. We demonstrate how to effectively overcome particle orientation bias by altering complex composition. In several of our examples, we use glycan density to facilitate interpretation of low-resolution 3D reconstruction and epitope mapping. Protein information plays an important role in our cryo-EM projects, especially in cases where we see significant challenges in obtaining high-resolution 3D maps.

scholarly journals Development of an integrated structural biology platform specialized for sub-100 kDa protein complexes to support biologics discovery and rational engineering

2021 ◽  
Author(s):  
Yuri Iozzo ◽  
Yu Qiu ◽  
Albert Xu ◽  
Anna Park ◽  
Maria Wendt ◽  
...  
Keyword(s):  
Decision Making ◽  
Structural Biology ◽  
Structural Information ◽  
Protein Complexes ◽  
Rapid Development ◽  
Protein Structures ◽  
Cost Effective ◽  
Particle Orientation ◽  
Dynamics Simulation ◽  
Rational Engineering

Developing a biologic medicine requires successful decision making at each step of selection, optimization, and/or combination of the right candidates at early research stages. Knowing the structural information and binding pattern between drug target and discovery candidates greatly increases the possibility of success. With the cryo-EM resolution revolution and rapid development of computational software, we have evaluated and integrated different tools in structural biology and the computation field and established a highly cost-effective platform, which allows us to obtain fast and accurate structural information for biologics projects with a close to 100% success rate and as fast as weeks turn-around time. Here we report four case studies selected from over 40 different protein structures and share how we integrate cryo-EM structure determination, computational structure modeling, and molecular dynamics simulation. With proper decision making and strategic planning, the platform allows us to obtain quality results within days to weeks, including sub-100 kDa complexes which are usually considered as a challenge due to their small size. Our utilization of this differential approach and use of multiple software packages, allows to manage priorities and resources to achieve goals quickly and efficiently. We demonstrate how to effectively overcome particle orientation bias by altering complex composition. In several of our examples, we use glycan density to facilitate interpretation of low-resolution 3D reconstruction and epitope mapping. Protein information plays an important role in our cryo-EM projects, especially in cases where we see significant challenges in obtaining high-resolution 3D maps.

scholarly journals Studying the Structures of Relaxed and Fuzzy Interactions: The Diverse World of S100 Complexes

2021 ◽  
Vol 8 ◽  
Author(s):  
Péter Ecsédi ◽  
Gergő Gógl ◽  
László Nyitray
Keyword(s):  
Neurodegenerative Diseases ◽  
Structural Biology ◽  
Binding Proteins ◽  
Structural Information ◽  
Protein Complexes ◽  
Point Of View ◽  
S100 Proteins ◽  
Current State ◽  
Diverse World

S100 proteins are small, dimeric, Ca2+-binding proteins of considerable interest due to their associations with cancer and rheumatic and neurodegenerative diseases. They control the functions of numerous proteins by forming protein–protein complexes with them. Several of these complexes were found to display “fuzzy” properties. Examining these highly flexible interactions, however, is a difficult task, especially from a structural biology point of view. Here, we summarize the available in vitro techniques that can be deployed to obtain structural information about these dynamic complexes. We also review the current state of knowledge about the structures of S100 complexes, focusing on their often-asymmetric nature.

scholarly journals Predicting the Shapes of Protein Complexes Through Collision Cross Section Measurements and Database Searches

2020 ◽  
Author(s):  
Michael Landreh ◽  
Cagla Sahin ◽  
Joseph Gault ◽  
Samira Sadeghi ◽  
Chester Lee Drum ◽  
...  
Keyword(s):  
Structural Biology ◽  
Cross Sections ◽  
Ion Mobility ◽  
Collision Cross Section ◽  
Protein Complexes ◽  
Protein Structures ◽  
Data Bank ◽  
Integral Membrane Protein ◽  
Specific Reference ◽  
Ion Mobility Mass Spectrometry

In structural biology, collision cross sections (CCS) from ion mobility mass spectrometry (IM-MS) measurements are routinely compared to computationally or experimentally derived protein structures. Here, we investigate whether CCS data can inform about the shape of a protein in the absence of specific reference structures. Analysis of the proteins in the CCS database shows that protein complexes with low apparent densities are structurally more diverse than those with a high apparent density. Using the CCS, molecular weight, and oligomeric states to mine the Protein Data Bank (PDB) for potentially similar protein structures, we find that we can distinguish oblate- and prolate-shaped protein complexes. We then apply the strategy to an integral membrane protein by comparing the shapes of a prokaryotic and an eukaryotic sodium/proton antiporter homologue. We conclude that mining the PDB with IM-MS data is a time-effective way to derive low-resolution structural models.

scholarly journals The expanding toolkit for structural biology: synchrotrons, X-ray lasers and cryoEM

IUCrJ ◽  
2019 ◽  
Vol 6 (2) ◽  
pp. 167-177 ◽  
Author(s):  
Stephen P. Muench ◽  
Svetlana V. Antonyuk ◽  
S. Samar Hasnain
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Structural Biology ◽  
Free Electron ◽  
Structural Information ◽  
Protein Complexes ◽  
Short Review ◽  
X Rays ◽  
Free Electron Lasers ◽  
X Ray

Structural biology continues to benefit from an expanding toolkit, which is helping to gain unprecedented insight into the assembly and organization of multi-protein machineries, enzyme mechanisms and ligand/inhibitor binding. The combination of results from X-ray free-electron lasers (XFELs), modern synchrotron crystallographic beamlines and cryo-electron microscopy (cryoEM) is proving to be particularly powerful. The highly brilliant undulator beamlines at modern synchrotron facilities have empowered the crystallographic revolution of high-throughput structure determination at high resolution. The brilliance of the X-rays at these crystallographic beamlines has enabled this to be achieved using microcrystals, but at the expense of an increased absorbed X-ray dose and a consequent vulnerability to radiation-induced changes. The advent of serial femtosecond crystallography (SFX) with X-ray free-electron lasers provides a new opportunity in which damage-free structures can be obtained from much smaller crystals (2 µm) and more complex macromolecules, including membrane proteins and multi-protein complexes. For redox enzymes, SFX provides a unique opportunity by providing damage-free structures at both cryogenic and ambient temperatures. The promise of being able to visualize macromolecular structures and complexes at high resolution without the need for crystals using X-rays has remained a dream, but recent technological advancements in cryoEM have made this come true and hardly a month goes by when the structure of a new/novel macromolecular assembly is not revealed. The uniqueness of cryoEM in providing structural information for multi-protein complexes, particularly membrane proteins, has been demonstrated by examples such as respirasomes. The synergistic use of cryoEM and crystallography in lead-compound optimization is highlighted by the example of the visualization of antimalarial compounds in cytochromebc1. In this short review, using some recent examples including our own work, we share the excitement of these powerful structural biology methods.

scholarly journals PhoX – an IMAC-enrichable Crosslinking Reagent

2019 ◽  
Author(s):  
Barbara A. Steigenberger ◽  
Roland J. Pieters ◽  
Albert J.R. Heck ◽  
Richard A. Scheltema
Keyword(s):  
Mass Spectrometry ◽  
Ribosomal Proteins ◽  
Structural Model ◽  
Structural Information ◽  
Protein Complexes ◽  
Protein Structures ◽  
Measurement Time ◽  
Close Proximity ◽  
Stoichiometric Reaction ◽  
Crosslinking Reagent

AbstractChemical crosslinking mass spectrometry is rapidly emerging as a prominent technique to study protein structures. Structural information is obtained by covalently connecting peptides in close proximity by small reagents and identifying the resulting peptide pairs by mass spectrometry. However, sub-stoichiometric reaction efficiencies render routine detection of crosslinked peptides problematic. Here we present a new tri-functional crosslinking reagent, termed PhoX, which is decorated with a stable phosphonic acid handle. This makes the crosslinked peptides amenable to the well-established IMAC enrichment. The handle allows for 300x enrichment efficiency and 97% specificity, dramatically reducing measurement time and improving data quality. We exemplify the approach on various model proteins and protein complexes, e.g. resulting in a structural model of the LRP1/RAP complex. PhoX is also applicable to whole cell lysates. When focusing the database search on ribosomal proteins, our first attempt resulted in 355 crosslinks, out-performing current efforts in less measurement time.

scholarly journals Predicting the Shapes of Protein Complexes Through Collision Cross Section Measurements and Database Searches

2020 ◽  
Author(s):  
Michael Landreh ◽  
Cagla Sahin ◽  
Joseph Gault ◽  
Samira Sadeghi ◽  
Chester Lee Drum ◽  
...  
Keyword(s):  
Structural Biology ◽  
Cross Sections ◽  
Ion Mobility ◽  
Collision Cross Section ◽  
Protein Complexes ◽  
Protein Structures ◽  
Data Bank ◽  
Integral Membrane Protein ◽  
Specific Reference ◽  
Ion Mobility Mass Spectrometry

In structural biology, collision cross sections (CCS) from ion mobility mass spectrometry (IM-MS) measurements are routinely compared to computationally or experimentally derived protein structures. Here, we investigate whether CCS data can inform about the shape of a protein in the absence of specific reference structures. Analysis of the proteins in the CCS database shows that protein complexes with low apparent densities are structurally more diverse than those with a high apparent density. Using the CCS, molecular weight, and oligomeric states to mine the Protein Data Bank (PDB) for potentially similar protein structures, we find that we can distinguish oblate- and prolate-shaped protein complexes. We then apply the strategy to an integral membrane protein by comparing the shapes of a prokaryotic and an eukaryotic sodium/proton antiporter homologue. We conclude that mining the PDB with IM-MS data is a time-effective way to derive low-resolution structural models.

Improved Sampling Strategies for Protein Model Refinement based on Molecular Dynamics Simulation

2020 ◽  
Author(s):  
Lim Heo ◽  
Collin Arbour ◽  
Michael Feig
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulation ◽  
Structure Prediction ◽  
Protein Structures ◽  
Conformational Space ◽  
Dynamics Simulation ◽  
Model Refinement ◽  
Protein Model ◽  
Lower Accuracy ◽  
Simulation Based

Protein structures provide valuable information for understanding biological processes. Protein structures can be determined by experimental methods such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or cryogenic electron microscopy. As an alternative, in silico methods can be used to predict protein structures. Those methods utilize protein structure databases for structure prediction via template-based modeling or for training machine-learning models to generate predictions. Structure prediction for proteins distant from proteins with known structures often results in lower accuracy with respect to the true physiological structures. Physics-based protein model refinement methods can be applied to improve model accuracy in the predicted models. Refinement methods rely on conformational sampling around the predicted structures, and if structures closer to the native states are sampled, improvements in the model quality become possible. Molecular dynamics simulations have been especially successful for improving model qualities but although consistent refinement can be achieved, the improvements in model qualities are still moderate. To extend the refinement performance of a simulation-based protocol, we explored new schemes that focus on an optimized use of biasing functions and the application of increased simulation temperatures. In addition, we tested the use of alternative initial models so that the simulations can explore conformational space more broadly. Based on the insight of this analysis we are proposing a new refinement protocol that significantly outperformed previous state-of-the-art molecular dynamics simulation-based protocols in the benchmark tests described here. <br>

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Protein Interaction Domains and Post-Translational Modifications: Structural Features and Drug Discovery Applications

2020 ◽  
Vol 27 (37) ◽  
pp. 6306-6355 ◽  
Author(s):  
Marian Vincenzi ◽  
Flavia Anna Mercurio ◽  
Marilisa Leone
Keyword(s):  
Drug Discovery ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Structural Information ◽  
Protein Complexes ◽  
Structural Features ◽  
Protein Protein Interactions ◽  
Modular Architecture ◽  
Post Translational Modifications ◽  
Interaction Domains

Background:: Many pathways regarding healthy cells and/or linked to diseases onset and progression depend on large assemblies including multi-protein complexes. Protein-protein interactions may occur through a vast array of modules known as protein interaction domains (PIDs). Objective:: This review concerns with PIDs recognizing post-translationally modified peptide sequences and intends to provide the scientific community with state of art knowledge on their 3D structures, binding topologies and potential applications in the drug discovery field. Method:: Several databases, such as the Pfam (Protein family), the SMART (Simple Modular Architecture Research Tool) and the PDB (Protein Data Bank), were searched to look for different domain families and gain structural information on protein complexes in which particular PIDs are involved. Recent literature on PIDs and related drug discovery campaigns was retrieved through Pubmed and analyzed. Results and Conclusion:: PIDs are rather versatile as concerning their binding preferences. Many of them recognize specifically only determined amino acid stretches with post-translational modifications, a few others are able to interact with several post-translationally modified sequences or with unmodified ones. Many PIDs can be linked to different diseases including cancer. The tremendous amount of available structural data led to the structure-based design of several molecules targeting protein-protein interactions mediated by PIDs, including peptides, peptidomimetics and small compounds. More studies are needed to fully role out, among different families, PIDs that can be considered reliable therapeutic targets, however, attacking PIDs rather than catalytic domains of a particular protein may represent a route to obtain selective inhibitors.

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