Professor Stephen Tucker

Abstract:

Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter containing a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two immunoglobulin-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophysical studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the site of peptide transport in eukaryotic cells.

Abstract:

Cerebral palsy is a sporadic disorder with multiple likely aetiologies, but frequently considered to be caused by birth asphyxia. Genetic investigations are rarely performed in patients with cerebral palsy and there is little proven evidence of genetic causes. As part of a large project investigating children with ataxia, we identified four patients in our cohort with a diagnosis of ataxic cerebral palsy. They were investigated using either targeted next generation sequencing or trio-based exome sequencing and were found to have mutations in three different genes, KCNC3, ITPR1 and SPTBN2. All the mutations were de novo and associated with increased paternal age. The mutations were shown to be pathogenic using a combination of bioinformatics analysis and in vitro model systems. This work is the first to report that the ataxic subtype of cerebral palsy can be caused by de novo dominant point mutations, which explains the sporadic nature of these cases. We conclude that at least some subtypes of cerebral palsy may be caused by de novo genetic mutations and patients with a clinical diagnosis of cerebral palsy should be genetically investigated before causation is ascribed to perinatal asphyxia or other aetiologies.

Abstract:

TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.