Solution NMR Approaches for Establishing Specificity of Weak Heterodimerization of Membrane Proteins

Abstract

Solution NMR provides a powerful approach for detecting complex formation involving weak to moderate intermolecular affinity. However, solution NMR has only rarely been used to detect complex formation between two membrane proteins in model membranes. The impact of specific binding on the NMR spectrum of a membrane protein can be difficult to distinguish from spectral changes that are induced by non-specific binding and/or by changes that arise from forced co-habitation of the two proteins in a single model membrane assembly. This is particularly the case when solubility limits make it impossible to complete a titration to the point of near-saturation of complex formation. In this work experiments are presented that provide the basis for establishing whether specific complex formation occurs between two membrane proteins under conditions where binding is not of high avidity. Application of these methods led to the conclusion that the membrane protein CD147 (also known as EMMPRIN or basigin) forms a specific heterodimeric complex in the membrane with the 99 residue transmembrane C-terminal fragment of the amyloid precursor protein (C99 or APP-βCTF), the latter being the immediate precursor of the amyloid-β polypeptides that are closely linked to the etiology of Alzheimer’s disease.

Soft matter perspective on protein crystal assembly

“…In biophysics, the distinction between specific and non-specific interactions typically relies on the existence of an energy gap that clearly divides a single, strong (specific) interaction from the other (nonspecific) ones [28,171]. In molecular biology, specific interactions are deemed responsible for the stoichiometric recognition of a given target, while non-specific interactions arise from the promiscuous yet non-biologically relevant association of molecules [94,95,172,173]. Specific interactions have thus been evolutionarily tuned to be free-energetically strong and geometrically oriented, while non-specific attractions have not.…”

Understanding single‐pass transmembrane receptor signaling from a structural viewpoint—what are we missing?

“…This is essentially analogous to the common approach of diluting a protein complex with the purpose of pushing the equilibrium toward the monomer conformation to allow its dissociation constant to be determined. In addition to GpA, monomer-dimer transitions as a result of changes in the DP ratio have been reported for many of the TMDs of which structures have been reported, e.g., the homodimers of FGFR3, VEGFR, ErbB1-4, APP, and TLR3 [23,[59][60][61][62][63]69,71,130].…”

Structural Insight into the Transmembrane Domain and the Juxtamembrane Region of the Erythropoietin Receptor in Micelles

“…S4). This result suggested that these residues might be important for dimerization if the hEpoR forms dimers when the detergent/ protein ratio is decreased, but it has to be noted that the line-broadening of crosspeaks may also be due to nonspecific dimerization or aggregation (43). Although several studies have been conducted to elucidate the TM-TM interface of the TMD of the EpoR, the TM dimer structure is not available.…”