Interaction of Substrates with γ-Secretase at the Level of Individual Transmembrane Helices-A Methodological Approach.

Intramembrane proteases, such as γ secretase, typically recruit multiple substrates from an excess of single-span membrane proteins. It is currently unclear to which extent substrate recognition depends on specific interactions of their transmembrane domains (TMDs) with TMDs of a protease. Here, we investigated a large number of potential pairwise interactions between TMDs of γ secretase and a diverse set of its substrates using two different configurations of BLaTM, a genetic reporter system. Our results reveal significant interactions between TMD2 of presenilin, the enzymatic subunit of γ secretase, and the TMD of the amyloid precursor protein, as well as of several other substrates. Presenilin TMD2 is a prime candidate for substrate recruitment, as has been shown from previous studies. In addition, the amyloid precursor protein TMD enters interactions with presenilin TMD 4 as well as with the TMD of nicastrin. Interestingly, the Gly-rich interfaces between the amyloid precursor protein TMD and presenilin TMDs 2 and 4 are highly similar to its homodimerization interface. In terms of methodology, the economics of the newly developed library-based method could prove to be a useful feature in related future work for identifying heterotypic TMD-TMD interactions within other biological contexts.

BLaTM 2.0, a Genetic Tool Revealing Preferred Antiparallel Interaction of Transmembrane Helix 4 of the Dual-Topology Protein EmrE.

Parallel and antiparallel transmembrane helix-helix interactions support the folding and non-covalent assembly of many integral membrane proteins. While several genetic tools are currently in use to study parallel transmembrane helix-helix interactions, antiparallel associations have been difficult to determine. Here, we present a novel genetic approach, termed BLaTM 2.0, which can be used in combination with the recently presented BLaTM 1.2 to compare the efficiency of antiparallel and parallel transmembrane domain (TMD) interactions in a natural membrane. In a practical application of the BLaTM system, we find that the antiparallel interaction of TMD4, the known dimerization domain of the dual-topology small multidrug transporter EmrE, is sequence-specific and much stronger than the parallel one. This suggests that TMD4 has evolved to favor the formation of dual-topology EmrE dimers over single-topology dimers.

Homodimerization Protects the Amyloid Precursor Protein C99 Fragment from Cleavage by γ-Secretase.

The amyloid precursor protein (APP) is a single-span integral membrane protein whose C-terminal fragment C99 is cleaved within the transmembrane helix by γ-secretase. Cleavage produces various Aß peptides that are linked to the etiology of Alzheimer's disease. The transmembrane helix is known to homodimerize in a sequence-specific manner, and considerable controversy about whether the homodimeric form of C99 is cleaved by γ-secretase exists. Here, we generated various covalent C99 homodimers via cross-linking at engineered cysteine residues. None of the homodimers was cleaved in vitro by purified γ-secretase, strongly suggesting that homodimerization protects C99 from cleavage.

The pathogenic A391E mutation in FGFR3 induces a structural change in the transmembrane domain dimer.

Fibroblast growth factor receptor 3 (FGFR3) is a single-pass membrane protein and a member of the receptor tyrosine kinase family of proteins that is involved in the regulation of skeletal growth and development. FGFR3 has three distinct domains: the ligand binding extracellular domain, the cytosolic kinase domain and the transmembrane domain (TMD). Previous work with the isolated FGFR3 TMD has shown that it has the ability to dimerize. Clinical and genetic studies have also correlated mutations in the TMD with a variety of skeletal and cranial dysplasias and cancer. Although the structures of the extracellular and cytosolic domains of FGFR3 have been solved, the structure of the TMD dimer is still unknown. Furthermore, very little is known regarding the effects of pathogenic mutations on the TMD dimer structure. We, therefore, carried out ToxR activity assays to determine the role of the SmXXXSm motif in the dimerization of the FGFR3 TMD. This motif has been shown to drive the association of many transmembrane proteins. Our results indicate that the interaction between wild-type FGFR3 TMDs is not mediated by two adjacent SmXXXSm motifs. In contrast, studies using the TMD carrying the pathogenic A391E mutation suggest that the motifs play a role in the dimerization of the mutant TMD. Based on these observations, here we report a new mechanistic model in which the pathogenic A391E mutation induces a structural change that leads to the formation of a more stable dimer.