Biology subjects

Kastritis, P. L.

Publications and source records attributed to Kastritis, P. L..

22 records · Page 2Linked to original sources

bioRxiv2020

Insights on cross-species transmission of SARS-CoV-2 from structural modeling

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global pandemic that has infected more than 14 million people in more than 180 countries worldwide. Like other coronaviruses, SARS-CoV-2 is thought to have been transmitted to humans from wild animals. Given the scale and widespread geographical distribution of the current pandemic, the question emerges whether human-to-animal transmission is possible and if so, which animal species are most at risk. Here, we investigated the structural properties of several ACE2 orthologs bound to the SARS-CoV-2 spike protein. We found that species known not to be susceptible to SARS-CoV-2 infection have non-conservative mutations in several ACE2 amino acid residues that disrupt key polar and charged contacts with the viral spike protein. Our models also predict affinity-enhancing mutations that could be used to design ACE2 variants for therapeutic purposes. Finally, our study provides a blueprint for modeling viral-host protein interactions and highlights several important considerations when designing these computational studies and analyzing their results.

bioinformatics↗

bioRxiv2020

Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

The ribosome is not only a highly complex molecular machine that executes translation according to the central dogma of molecular biology, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker diacetyl dibutyric urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all cross-links, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.

biochemistry↗

bioRxiv2019

Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin receptor assemblies

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their ubiquitylation targets, AUX/IAAs, sense auxin concentrations in the nucleus. TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, we resolved TIR1{middle dot}auxin{middle dot}IAA7 and TIR1{middle dot}auxin{middle dot}IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degrons vicinity, cooperatively position AUX/IAAs on TIR1. The AUX/IAA PB1 interaction domain also assists in non-native contacts, affecting AUX/IAA dynamic interaction states. Our results establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for adopting a multiplicity of functional states. We postulate IDRs in distinct members of the AUX/IAA family to be an adaptive signature for protein interaction and initiation region for proteasome recruitment.

plant biology↗

bioRxiv2019

2.7 A cryo-EM structure of vitrified M. musculus H-chain apoferritin from 200 keV \"screening microscope\"

Here we present the structure of mouse H-chain apoferritin at 2.7 [A] (FSC=0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device. Data were collected using a compact, two-lens illumination system with a constant power objective lens, without the use of energy filters or aberration correctors. Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (x-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving resolution close to the Rayleigh limit. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging, widely thought of as a "screening cryo-microscope", can be used to determine structures approaching atomic resolution. HighlightsO_LIThe 2.7 [A] structure of mouse apoferritin was solved using a 200 keV screening cryo-microscope C_LIO_LIThe apoferritin reconstruction was resolved without an energy filter, aberration correctors, or constant-power condenser lenses C_LIO_LIComparison to available crystallographic and cryo-EM structures from high-end cryo-microscopes demonstrates consistency in resolved water molecules, metals and side chain orientations C_LIO_LIAlthough radiation damage is more prominent at 200 keV compared to 300 keV, this type of instrumentation is more accessible to research laboratories due to its compactness and simplicity C_LI

biophysics↗