Structural basis for the recognition of the bacterial tyrosine kinase Wzc by its cognate tyrosine phosphatase Wzb.

Bacterial tyrosine kinases (BY-kinases) comprise a family of protein tyrosine kinases that are structurally distinct from their functional counterparts in eukaryotes and are highly conserved across the bacterial kingdom. BY-kinases act in concert with their counteracting phosphatases to regulate a variety of cellular processes, most notably the synthesis and export of polysaccharides involved in biofilm and capsule biogenesis. Biochemical data suggest that BY-kinase function involves the cyclic assembly and disassembly of oligomeric states coupled to the overall phosphorylation levels of a C-terminal tyrosine cluster. This process is driven by the opposing effects of intermolecular autophosphorylation, and dephosphorylation catalyzed by tyrosine phosphatases. In the absence of structural insight into the interactions between a BY-kinase and its phosphatase partner in atomic detail, the precise mechanism of this regulatory process has remained poorly defined. To address this gap in knowledge, we have determined the structure of the transiently assembled complex between the catalytic core of the Escherichia coli (K-12) BY-kinase Wzc and its counteracting low-molecular weight protein tyrosine phosphatase (LMW-PTP) Wzb using solution NMR techniques. Unambiguous distance restraints from paramagnetic relaxation effects were supplemented with ambiguous interaction restraints from static spectral perturbations and transient chemical shift changes inferred from relaxation dispersion measurements and used in a computational docking protocol for structure determination. This structurepresents an atomic picture of the mode of interaction between an LMW-PTP and its BY-kinase substrate, and provides mechanistic insight into the phosphorylation-coupled assembly/disassembly process proposed to drive BY-kinase function.

Molecular determinants and thermodynamics of the amyloid precursor protein transmembrane domain implicated in Alzheimer's disease.

The deposition of toxic amyloid-ß (Aß) peptide aggregates in the brain is a hallmark of Alzheimer's disease. The intramembrane proteolysis by γ-secretase of the amyloid precursor protein ß-carboxy-terminal fragment (APP-ßCTF) constitutes the final step in the production of Aß peptides. Mounting evidence suggests that APP-ßCTF is a transmembrane domain (TMD) dimer, and that dimerization might modulate the production of Aß species that are prone to aggregation and are therefore most toxic. We combined experimental and computational approaches to study the molecular determinants and thermodynamics of APP-ßCTF dimerization, and we produced a unifying structural model that reconciles much of the published data. Using a cell assay that exploits a dimerization-dependent activator of transcription, we identified specific dimerization-affecting mutations located mostly at the N-terminus of the TMD of APP-ßCTF. The ability of selected mutants to affect the dimerization of full-length APP-ßCTF was confirmed by fluorescence resonance energy transfer experiments. Free-energy estimates of the wild type and mutants of the TMD of APP-ßCTF derived from enhanced molecular dynamics simulations showed that the dimeric state is composed of different arrangements, in which either (709)GXXXA(713) or (700)GXXXG(704)GXXXG(708) interaction motifs can engage in symmetric or asymmetric associations. Mutations along the TMD of APP-ßCTF were found to modulate the relative free energy of the dimeric configurations and to differently affect the distribution of interfaces within the dimeric state. This observation might have important biological implications, since dimers with a different arrangement of the transmembrane helices are likely to be recognized differently by γ-secretase and to lead to a variation in Aß levels.