Oligomerization-driven MLKL ubiquitylation antagonises necroptosis

Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor Interacting serine/threonine Protein Kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor significance of this event have been demonstrated. Here we show that necroptosis-specific, multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin insoluble cell fraction comprising plasma/organellar membranes and protein aggregates. This ubiquitylated form is diminished by a plasma membrane located deubiquitylating enzyme. MLKL is ubiquitylated on at least 4 separate lysine residues once oligomerized, and this correlates with proteasome- and lysosome- dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licenses MLKL auto-activity independent of necroptosis signalling in mouse and human cells. Therefore, besides its role in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, ubiquitylation also contributes to the restraint of basal levels of activated MLKL to avoid errant cell death.

Structural comparison of GLUT1 to GLUT3 reveal transport regulation mechanism in sugar porter family

The human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here, we present a 2.4 Å crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic “SP motif” and a conserved “A motif” stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl− ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the SP family.

On-rate modulation of cadherin interactions by chemical fragments

Many cadherin family proteins are associated with diseases such as cancer. Since cell adhesion requires homodimerization of cadherin molecules, a small-molecule regulator of dimerization would have therapeutic potential. Herein, we describe identification of a P-cadherin-specific chemical fragment that inhibits P-cadherin-mediated cell adhesion. Although the identified molecule is a fragment compound, it binds to a cavity of P-cadherin that has not previously been targeted, indirectly prevents formation of hydrogen bonds necessary for formation of an intermediate called the X dimer and thus modulates the on-rate of X dimerization. Our findings will impact on a strategy for kinetic regulation of protein-protein interactions and stepwise assembly of protein complexes using small molecules.

Mechanics and kinetics of dynamic instability

During dynamic instability, self-assembling microtubules (MTs) stochastically alternate between phases of growth and shrinkage. This process is driven by the presence of two distinct states of MT subunits, GTP- and GDP-bound tubulin dimers, that have different structural properties. Here, we use a combination of analysis and computer simulations to study the mechanical and kinetic regulation of dynamic instability in three-dimensional (3D) self-assembling MTs. Our model quantifies how the 3D structure and kinetics of the distinct states of tubulin dimers determine the mechanical stability of MTs. We further show that dynamic instability is influenced by the presence of quenched disorder in the state of the tubulin subunit as reflected in the fraction of non-hydrolysed tubulin. Our results connect the 3D geometry, kinetics and statistical mechanics of these tubular assemblies within a single framework, and may be applicable to other self-assembled systems where these same processes are at play.

Structural comparison of GLUT1 to GLUT3 reveal transport regulation mechanism in Sugar Porter family

The human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here we present a 2.4 Å crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic “Sugar Porter motif” and a conserved “A motif” stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl- ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the Sugar Porter family.Summary BlurbNew structure of GLUT1 compared to GLUT3 explain different substrate affinities. The result provide a functional rationale for key structural motifs that define the universal Sugar Porter family.