Structure and conformational variability of the HER2-trastuzumab-pertuzumab complex.

Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we obtain the detailed structure of a particularly difficult complex between human epidermal growth factor receptor 2 (HER2) and the antigen-binding fragments from two distinct therapeutic antibodies binding to distant parts of the flexible HER2, pertuzumab and trastuzumab (HTP). We highlight the strengths and limitations of current data processing software in dealing with various kinds of heterogeneities, particularly continuous conformational heterogeneity, and in describing the motions that can be extracted from our dataset. Our HTP structure provides a more detailed view than the one previously available for this ternary complex. This allowed us to pinpoint a previously overlooked loop in domain IV that may be involved both in binding of trastuzumab and in HER2 dimerization. This finding may contribute to explain the synergistic anticancer effect of the two antibodies. We further propose that the flexibility of the HTP complex, beyond the difficulties it causes for cryo-EM analysis, actually reflects regulation of HER2 signaling and its inhibition by therapeutic antibodies. Notably we obtain our best data with ultra-thin continuous carbon grids, showing that with current cameras their use to alleviate particle misdistribution is compatible with a protein complex of only 162 kDa. Perhaps most importantly, we provide here a dataset for such a smallish protein complex for further development of software accounting for continuous conformational heterogeneity in cryo-EM images.

Analysis of the Conformational Landscape of the N-Domains of the AAA ATPase p97: Disentangling the Continuous Conformational Variability in Partially Symmetrical Complexes.

Single-particle cryo-electron microscopy (cryo-EM) has been shown to be effective in defining the structure of macromolecules, including protein complexes. Complexes adopt different conformations and compositions to perform their biological functions. In cryo-EM, the protein complexes are observed in solution, enabling the recording of images of the protein in multiple conformations. Various methods exist for capturing the conformational variability through analysis of cryo-EM data. Here, we analyzed the conformational variability in the hexameric AAA + ATPase p97, a complex with a six-fold rotational symmetric core surrounded by six flexible N-domains. We compared the performance of discrete classification methods with our recently developed method, MDSPACE, which uses 3D-to-2D flexible fitting of an atomic structure to images based on molecular dynamics (MD) simulations. Our analysis detected a novel conformation adopted by approximately 2% of the particles in the dataset and determined that the N-domains of p97 sway by up to 60° around a central position. This study demonstrates the application of MDSPACE in analyzing the continuous conformational changes in partially symmetrical protein complexes, systems notoriously difficult to analyze due to the alignment errors caused by their partial symmetry.

MDTOMO method for continuous conformational variability analysis in cryo electron subtomograms based on molecular dynamics simulations.

Cryo electron tomography (cryo-ET) allows observing macromolecular complexes in their native environment. The common routine of subtomogram averaging (STA) allows obtaining the three-dimensional (3D) structure of abundant macromolecular complexes, and can be coupled with discrete classification to reveal conformational heterogeneity of the sample. However, the number of complexes extracted from cryo-ET data is usually small, which restricts the discrete-classification results to a small number of enough populated states and, thus, results in a largely incomplete conformational landscape. Alternative approaches are currently being investigated to explore the continuity of the conformational landscapes that in situ cryo-ET studies could provide. In this article, we present MDTOMO, a method for analyzing continuous conformational variability in cryo-ET subtomograms based on Molecular Dynamics (MD) simulations. MDTOMO allows obtaining an atomic-scale model of conformational variability and the corresponding free-energy landscape, from a given set of cryo-ET subtomograms. The article presents the performance of MDTOMO on a synthetic ABC exporter dataset and an in situ SARS-CoV-2 spike dataset. MDTOMO allows analyzing dynamic properties of molecular complexes to understand their biological functions, which could also be useful for structure-based drug discovery.

MDSPACE: Extracting Continuous Conformational Landscapes from Cryo-EM Single Particle Datasets Using 3D-to-2D Flexible Fitting based on Molecular Dynamics Simulation.

This article presents an original approach for extracting atomic-resolution landscapes of continuous conformational variability of biomolecular complexes from cryo electron microscopy (cryo-EM) single particle images. This approach is based on a new 3D-to-2D flexible fitting method, which uses molecular dynamics (MD) simulation and is embedded in an iterative conformational-landscape refinement scheme. This new approach is referred to as MDSPACE, which stands for Molecular Dynamics simulation for Single Particle Analysis of Continuous Conformational hEterogeneity. The article describes the MDSPACE approach and shows its performance using synthetic and experimental datasets.

MDSPACE and MDTOMO Software for Extracting Continuous Conformational Landscapes from Datasets of Single Particle Images and Subtomograms Based on Molecular Dynamics Simulations: Latest Developments in ContinuousFlex Software Package.

Cryo electron microscopy (cryo-EM) instrumentation allows obtaining 3D reconstruction of the structure of biomolecular complexes in vitro (purified complexes studied by single particle analysis) and in situ (complexes studied in cells by cryo electron tomography). Standard cryo-EM approaches allow high-resolution reconstruction of only a few conformational states of a molecular complex, as they rely on data classification into a given number of classes to increase the resolution of the reconstruction from the most populated classes while discarding all other classes. Such discrete classification approaches result in a partial picture of the full conformational variability of the complex, due to continuous conformational transitions with many, uncountable intermediate states. In this article, we present the software with a user-friendly graphical interface for running two recently introduced methods, namely, MDSPACE and MDTOMO, to obtain continuous conformational landscapes of biomolecules by analyzing in vitro and in situ cryo-EM data (single particle images and subtomograms) based on molecular dynamics simulations of an available atomic model of one of the conformations. The MDSPACE and MDTOMO software is part of the open-source ContinuousFlex software package (starting from version 3.4.2 of ContinuousFlex), which can be run as a plugin of the Scipion software package (version 3.1 and later), broadly used in the cryo-EM field.

ContinuousFlex: Software package for analyzing continuous conformational variability of macromolecules in cryo electron microscopy and tomography data.

ContinuousFlex is a user-friendly open-source software package for analyzing continuous conformational variability of macromolecules in cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET) data. In 2019, ContinuousFlex became available as a plugin for Scipion, an image processing software package extensively used in the cryo-EM field. Currently, ContinuousFlex contains software for running (1) recently published methods HEMNMA-3D, TomoFlow, and NMMD; (2) earlier published methods HEMNMA and StructMap; and (3) methods for simulating cryo-EM and cryo-ET data with conformational variability and methods for data preprocessing. It also includes external software for molecular dynamics simulation (GENESIS) and normal mode analysis (ElNemo), used in some of the mentioned methods. The HEMNMA software has been presented in the past, but not the software of other methods. Besides, ContinuousFlex currently also offers a deep learning extension of HEMNMA, named DeepHEMNMA. In this article, we review these methods in the context of the ContinuousFlex package, developed to facilitate their use by the community.

In silico prediction, characterization, docking studies and molecular dynamics simulation of human p97 in complex with p37 cofactor.

BACKGROUND: The AAA + ATPase p97 is an essential unfoldase/segragase involved in a multitude of cellular processes. It functions as a molecular machine critical for protein homeostasis, homotypic membrane fusion events and organelle biogenesis during mitosis in which it acts in concert with cofactors p47 and p37. Cofactors assist p97 in extracting and unfolding protein substrates through ATP hydrolysis. In contrast to other p97's cofactors, p37 uniquely increases the ATPase activity of p97. Disease-causing mutations in p97, including mutations that cause neurodegenerative diseases, increase cofactor association with its N-domain, ATPase activity and improper substrate processing. Upregulation of p97 has also been observed in various cancers. This study aims towards the characterization of the protein-protein interaction between p97 and p37 at the atomic level. We defined the interacting residues in p97 and p37. The knowledge will facilitate the design of unique small molecules inhibiting this interaction with insights into cancer therapy and drug design. RESULTS: The homology model of human p37 UBX domain was built from the X-ray crystal structure of p47 C-terminus from rat (PDB code:1S3S, G) as a template and assessed by model validation analysis. According to the HDOCK, HAWKDOCK, MM-GBSA binding free energy calculations and Arpeggio, we found that there are several hydrophobic and two hydrogen-bonding interactions between p37 UBX and p97 N-D1 domain. Residues of p37 UBX predicted to be involved in the interactions with p97 N-D1 domain interface are highly conserved among UBX cofactors. CONCLUSION: This study provides a reliable structural insight into the p37-p97 complex binding sites at the atomic level though molecular docking coupled with molecular dynamics simulation. This can guide the rational design of small molecule drugs for inhibiting mutant p97 activity.

NMMD: Efficient Cryo-EM Flexible Fitting Based on Simultaneous Normal Mode and Molecular Dynamics atomic displacements.

Atomic models of cryo electron microscopy (cryo-EM) maps of biomolecular conformations are often obtained by flexible fitting of the maps with available atomic structures of other conformations (e.g., obtained by X-ray crystallography). This article presents a new flexible fitting method, NMMD, which combines normal mode analysis (NMA) and molecular dynamics simulation (MD). Given an atomic structure and a cryo-EM map to fit, NMMD simultaneously estimates global atomic displacements based on NMA and local displacements based on MD. NMMD was implemented by modifying EMfit, a flexible fitting method using MD only, in GENESIS 1.4. As EMfit, NMMD can be run with replica exchange umbrella sampling procedure. The new method was tested using a variety of EM maps (synthetic and experimental, with different noise levels and resolutions). The results of the tests show that adding normal modes to MD-based fitting makes the fitting faster (40% in average) and, in the majority of cases, more accurate.