Microsomal glutathione S-transferase 2 (MGST2) produces leukotriene C-4, key for intracrine signaling of endoplasmic reticulum (ER) stress, oxidative DNA damage and cell death. MGST2 trimer restricts catalysis to only one out of three active sites at a time, but the molecular basis is unknown. Here, we present crystal structures of human MGST2 combined with biochemical and computational evidence for a concerted mechanism, involving local unfolding coupled to global conformational changes that regulate catalysis. Furthermore, synchronized changes in the biconical central pore modulate the hydrophobicity and control solvent influx to optimize reaction conditions at the active site. These unique mechanistic insights pertain to other, structurally related, drug targets. Microsomal glutathione S-transferase 2 (MGST2) produces leukotriene C-4, an intracrine mediator of cell death. Structural, biochemical and computational analyses of human MGST2 suggest a mechanism employed by the enzyme to restrict catalysis to only one active site within the MGST2 trimer.

Na⁺/H⁺ exchangers (NHEs) are ancient membrane-bound nanoma- chines that work to regulate intracellular pH, sodium levels and cell volume. NHE activities contribute to the control of the cell cycle, cell proliferation, cell migration and vesicle trafficking. NHE dysfunction has been linked to many diseases, and they are targets of pharma- ceutical drugs. Despite their fundamental importance to cell home- ostasis and human physiology, structural information for the mammalian NHEs was lacking. Here, we report the cryogenic elec- tron microscopy structure of NHE isoform 9 (SLC9A9) from Equus caballus at 3.2 Å resolution, an endosomal isoform highly expressed in the brain and associated with autism spectrum (ASD) and atten- tion deficit hyperactivity (ADHD) disorders. Despite low sequence identity, the NHE9 architecture and ion-binding site are remarkably most similar to distantly related bacterial Na⁺/H⁺ antiporters with 13 transmembrane segments. Collectively, we reveal the conserved architecture of the NHE ion-binding site, their elevator-like structural transitions, the functional implications of autism disease mutations and the role of phosphoinositide lipids to promote homodimerization that, together, have important physiological ramifications.

Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists(1), the hexose transporter from the malaria parasite Plasmodium falciparum PfHT1(2,3) has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with d-glucose at a resolution of 3.6 angstrom. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures(4,5). Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 angstrom from d-glucose) are just as critical for transport as the residues that directly coordinate d-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics. Crystal structure of the Plasmodium falciparum hexose transporter PfHT1 reveals the molecular basis of its ability to transport multiple types of sugar as efficiently as the dedicated mammalian glucose and fructose transporters.

EGFR mutations display striking organ-site asymmetry and heterogeneity. We have shown that structurally diverse extracellular mutations, typical of glioblastomas, converge to a similar intermediate conformation, which can be synergistically targeted extra- and intracelullarly by antibody mAb806 and type-II kinase inhibitors. Our findings reveal convergence behind heterogeneity, paving the way for allostery-based co-targeting.

Understanding how proteins transition between different conformers, and how conformers relate to each other in terms of structure and function, is not trivial. Here, we present an online tool for transition pathway generation between two protein conformations using Elastic Network Driven Brownian Dynamics Importance Sampling, a coarse-grained simulation algorithm, which spontaneously predicts transition intermediates trapped experimentally. In addition to path-generation, the server provides an interactive 2D-motion landscape graphical representation of the transitions or any additional conformers to explore their structural relationships.

Ionizing radiation (IR) and chemotherapy are standard-of-care treatments for glioblastoma (GBM) patients and both result in DNA damage, however, the clinical efficacy is limited due to therapeutic resistance. We identified a mechanism of such resistance mediated by phosphorylation of PTEN on tyrosine 240 (pY240-PTEN) by FGFR2. pY240-PTEN is rapidly elevated and bound to chromatin through interaction with Ki-67 in response to IR treatment and facilitates the recruitment of RAD51 to promote DNA repair. Blocking Y240 phosphorylation confers radiation sensitivity to tumors and extends survival in GBM preclinical models. Y240F-Pten knockin mice showed radiation sensitivity. These results suggest that FGFR-mediated pY240-PTEN is a key mechanism of radiation resistance and is an actionable target for improving radiotherapy efficacy.

Epidermal growth factor receptor (EGFR) signaling is initiated by a large ligand-favored conformational change of the extracellular domain (ECD) from a closed, self-inhibited tethered monomer, to an open untethered state, which exposes a loop required for strong dimerization and activation. In glioblastomas (GBMs), structurally heterogeneous missense and deletion mutations concentrate at the ECD for unclear reasons. We explore the conformational impact of GBM missense mutations, combining elastic network models (ENMs) with multiple molecular dynamics (MD) trajectories. Our simulations reveal that the main missense class, located at the I-II interface away from the self-inhibitory tether, can unexpectedly favor spontaneous untethering to a compact intermediate state, here validated by small-angle X-ray scattering (SAXS). Significantly, such intermediate is characterized by the rotation of a large ECD fragment (N-TR1), deleted in the most common GBM mutation, EGFRvIII, and that makes accessible a cryptic epitope characteristic of cancer cells. This observation suggested potential structural equivalence of missense and deletion ECD changes in GBMs. Corroborating this hypothesis, our FACS, in vitro, and in vivo data demonstrate that entirely different ECD variants all converge to remove N-TR1 steric hindrance from the 806-epitope, which we show is allosterically coupled to an intermediate kinase and hallmarks increased oncogenicity. Finally, the detected extraintracellular coupling allows for synergistic cotargeting of the intermediate with mAb806 and inhibitors, which is proved herein.

We explored the clinical and pathological impact of epidermal growth factor receptor (EGFR) extracellular domain missense mutations. Retrospective assessment of 260 de novo glioblastoma patients revealed a significant reduction in overall survival of patients having tumors with EGFR mutations at alanine 289 (EGFR(A289D/T/V)). Quantitative multi-parametric magnetic resonance imaging analyses indicated increased tumor invasion for EGFR(A289D/T/V) mutants, corroborated in mice bearing intracranial tumors expressing EGFR(A289V) and dependent on ERK-mediated expression of matrix metalloproteinase-1. EGFR(A289V) tumor growth was attenuated with an antibody against a cryptic epitope, based on in silico simulation. The findings of this study indicate a highly invasive phenotype associated with the EGFR(A289V) mutation in glioblastoma, postulating EGFR(A289V) as a molecular marker for responsiveness to therapy with EGFR-targeting antibodies.