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An automated platform for structural analysis of membrane proteins through serial crystallography

ABSTRACT: Summary Membrane proteins are central to many pathophysiological processes, yet remain very difficult to analyze structurally. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperatures. We apply this approach to human integral membrane proteins, which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting, and serial crystallography, enabling screening of small-molecule libraries with membrane protein crystals grown in meso. This approach brings needed automation to this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins. Graphical abstract Highlights • A platform for rapid in meso structures by serial crystallography (SSX)• Insights into ADIPOR2 receptor-ligand dynamic interactions• A web-based application for remote user-guided experimental design and execution• An automated SSX-based ligand discovery pipeline for membrane proteins is introduced Motivation Membrane proteins are key regulators of most physiological processes and represent attractive targets for drug discovery programs. One of the most successful methods to obtain high-resolution structures of membrane proteins relies on in meso crystallization in combination with serial synchrotron crystallography. However, this remains a difficult process, with challenges at every step, including complex manual sample recovery protocols leading to limited throughput and sample loss and the difficulty in carrying out high-throughput ligand screening experiments. We have developed a new approach enabling rapid, automated structural analysis of membrane proteins in meso based on the CrystalDirect technology that addresses these issues, enabling high-throughput drug discovery with membrane proteins. Membrane proteins control many biological processes and represent attractive targets for drug discovery, but are difficult to study structurally. Healey et al. present an automated approach, combining the CrystalDirect technology and serial crystallography, for rapid structural analysis of membrane proteins and opening new opportunities for high-throughput drug discovery.

SUBMITTER: Healey R

PROVIDER: S-EPMC8545655 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins, including the β2-adrenoreceptor-Gs protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existing in meso crystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development.

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An automated platform for structural analysis of membrane proteins through serial crystallography

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