Complex Formation and Light Activation in Membrane-Embedded Sensory Rhodopsin II as Seen by Solid-State NMR Spectroscopy

Abstract

Microbial rhodopsins execute diverse biological functions in the cellular membrane. A mechanistic understanding of their functional profile is, however, still limited. We used solid-state NMR (ssNMR) spectroscopy to study structure and dynamics of a 2 x 400 amino acid sensory rhodopsin/transducer (SRII/HtrII) complex from Natronomonas pharaonis in a natural membrane environment. We found a receptor-transducer binding interface in the ground state that significantly extends beyond the available X-ray structure. This binding domain involves the EF loop of the receptor and stabilizes the functionally relevant, directly adjacent HAMP domain of the transducer. Using 2D ssNMR difference spectroscopy, we identified protein residues that may act as a functional module around the retinal binding site during the early events of protein activation. These latter protein segments, the inherent plasticity of the HAMP domain, and the observation of an extended SRII/HtrII membrane-embedded interface may be crucial components for optimal signal relay efficiency across the cell membrane.

Ground state structure of D75N mutant of sensory rhodopsin II in complex with its cognate transducer

“…7 D75N mutant are quite similar. Changes observed are consistent with a rearrangement of the hydrogen bonding network close to retinal Schiff base [28].…”

Active State of Sensory Rhodopsin II: Structural Determinants for Signal Transfer and Proton Pumping

“…They may result from the crystal packing (E-F loops of adjacent proteins form a crystal contact), or may be a genuine conformational change in the active state of uncomplexed NpSRII. It has been shown by solid state NMR spectroscopy, fluorescence and EPR spectroscopy that the E-F loop is interacting tightly with the transducer [38][39][40] . However, it is clear that the E-F loop is not crucial for signal transduction, as NpSRII E-F loop deletion mutants as well as BR triple mutant, which has a small disordered E-F loop, are all capable of signaling 20,41 .…”

Recent Solid-State NMR Studies of Membrane-Bound Peptides and Proteins

“…Adequate resolution of C-C correlations is key to successful structure determination experiment, because the 5-70 ppm aliphatic region is the fingerprint region for residue types. Well-resolved 13 C-13 C correlations have recently been reported for a number of membrane proteins, including the trimeric autotransporter adhesion protein YadA [170], the potassium channel KcsA-Kv1.3 [178], human aquaporin-1 [179], DsbB [172], proteorhodopsin [180,181], and sensory rhodopsin II (SRII) [182]. NCOCX (for inter-residue along protein backbone), NCACX (for intraresidue), and CONCA (for inter-residue) with 13 C-13 C correlations construct spin systems for individual amino acid residues and connect the spin systems along the amino acid sequence.…”