Bliven, S. | bioRxiv Science

bioRxiv2021

Cryo-EM structure of a single-chain β1-adrenoceptor - AmpC β-lactamase fusion protein

The insertion of fusion proteins has enabled the crystallization of a wide range of G-protein-coupled receptors. Here, we adapted this engineering strategy to cryo-electron microscopy (cryo-EM). We inserted the soluble protein AmpC {beta}-lactamase into the third intracellular loop (ICL3) of ultra-thermostable {beta}1-adrenoceptor ({beta}1AR) via chimeric helix fusions. Biochemical and biophysical characterization showed that the resulting fusion protein after expression, solubilization and purification was monodisperse and able to bind the known {beta}1AR weak partial agonist cyanopindolol, and the antagonist propranolol. The protein particles comprised sufficient mass and discernable structural features to elucidate its cryo-EM structure in complex with cyanopindolol without any natural (G-proteins, arrestins) or artificial (Nanobodies, DARPins) binding partners, to an overall resolution of 4.2 [A]. The seven-helix architecture and helix eight, as well as both GPCR - AmpC {beta}-lactamase connections are clearly resolved. {beta}1AR is in its inactive conformation. 3D variability analysis revealed significant flexibility between the two protein domains and within the GPCR helices, offering insights into conformational dynamics. The map contains clear density for the cyanopindolol. The fusion protein geometry theoretically fits a wide range of class A GPCRs, presenting a powerful platform for structure elucidation of a diverse array of class A GPCR - ligand complexes by cryo-EM in the inactive receptor state. The approach furthermore holds potential for structure elucidation of GPCRs in the absence of ligands.

molecular biology↗

bioRxiv2017

Automated evaluation of quaternary structures from protein crystals

A correct assessment of the quaternary structure of proteins is a fundamental prerequisite to understanding their function, physico-chemical properties and mode of interaction with other proteins. Currently about 90% of structures in the Protein Data Bank are crystal structures, in which the correct quaternary structure is embedded in the crystal lattice among a number of crystal contacts. Computational methods are required to 1) classify all protein-protein contacts in crystal lattices as biologically relevant or crystal contacts and 2) provide an assessment of how the biologically relevant interfaces combine into a biological assembly In our previous work we addressed the first problem with our EPPIC (Evolutionary Protein Protein Interface Classifier) method. Here, we present our solution to the second problem with a new method that combines the interface classification results with symmetry and topology considerations. The new algorithm enumerates all possible valid assemblies within the crystal using a graph representation of the lattice and predicts the most probable biological unit based on the pairwise interface scoring. Our method achieves 85% precision on a new dataset of 1,481 biological assemblies with consensus of PDB annotations. Although almost the same precision is achieved by PISA, currently the most popular quaternary structure assignment method, we show that, due to the fundamentally different approach to the problem, the two methods are complementary and could be combined to improve biological assembly assignments. The software for the automatic assessment of protein assemblies (EPPIC version 3) has been made available through a web server at http://www.eppic-web.org.\n\nAuthor summaryX-ray diffraction experiments are the main experimental technique to reveal the detailed atomic 3-dimensional structure of proteins. In these experiments, proteins are packed into crystals, an environment that is far away from their native solution environment. Determining which parts of the structure reflect the proteins state in the cell rather than being artifacts of the crystal environment can be a difficult task. How the different protein subunits assemble together in solution is known as the quaternary structure. Finding the correct quaternary structure is important both to understand protein oligomerization and for the understanding of protein-protein interactions at large. Here we present a new method to automatically determine the quaternary structure of proteins given their crystal structure. We provide a theoretical basis for properties that correct protein assemblies should possess, and provide a systematic evaluation of all possible assemblies according to these properties. The method provides a guidance to the experimental structural biologist as well as to structural bioinformaticians analyzing protein structures in bulk. Assemblies are provided for all proteins in the Protein Data Bank through a public website and database that is updated weekly as new structures are released.

bioinformatics↗