Structural Disorder of the CD3ζ Transmembrane Domain Studied with 2D IR Spectroscopy and Molecular Dynamics Simulations

Abstract

In a recently reported study [Mukherjee, et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3528], we used 2D IR spectroscopy and 1-13 C═ 18 O isotope labeling to measure the vibrational dynamics of 11 amide I modes in the CD3ζ transmembrane domain. We found that the homogeneous line widths and population relaxation times were all nearly identical, but that the amount of inhomogeneous broadening correlated with the position of the amide group inside the membrane. In this study, we use molecular dynamics simulations to investigate the structural and dynamical origins of these experimental observations. We use two models to convert the simulations to frequency trajectories from which the mean frequencies, standard deviations, frequency correlation functions, and 2D IR spectra are calculated. Model 1 correlates the hydrogen-bond length to the amide I frequency, whereas model 2 uses an ab initio-based electrostatic model. We find that the structural distributions of the peptidic groups and their environment are reflected in the vibrational dynamics of the amide I modes. Environmental forces from the water and lipid headgroups partially denature the helices, shifting the infrared frequencies and creating larger inhomogeneous distributions for residues near the ends. The least inhomogeneously broadened residues are those located in the middle of the membrane where environmental electrostatic forces are weakest and the helices are most ordered. Comparison of the simulations to experiment confirms that the amide I modes near the C-terminal are larger than at the N-terminal because of the asymmetric structure of the peptide bundle in the membrane. The comparison also reveals that residues at a kink in the α-helices have broader line widths than more helical parts of the peptide because the peptide backbone at the kink exhibits a larger amount of structural disorder. Taken together, the simulations and experiments reveal that infrared line shapes are sensitive probes of membrane protein structural and environmental heterogeneity.

The Optimal Antigen Response of Chimeric Antigen Receptors Harboring the CD3ζ Transmembrane Domain Is Dependent upon Incorporation of the Receptor into the Endogenous TCR/CD3 Complex

“…In fact, at transmembrane position 9, not only was dimerization abrogated but also the resulting receptor (C2G L9C) was markedly affected with regard to Ag sensitivity, suggesting this residue plays an important functional role in CD3z signaling. Data supporting this theory come from two-dimensional infrared spectroscopic analysis of the CD3z transmembrane domain in which a kink in the dimer at position L9 was identified (32,33). This kink may play a functional role in regulating the structural deformation or twisting/piston-like movement that is predicted to precede TCR signaling events (34)(35)(36).…”

Optimization of Model Parameters for Describing the Amide I Spectrum of a Large Set of Proteins

“…Parametrized electrostatic calculations using the building block approach have been used in protein amide I sim-ulations of ubiquitin, 10,33 other proteins 34 as well as polypeptides in membrane environment. 29,35 These calculations are generally combined with MD simulations that provide coordinate trajectories, which are then utilized for calculating instantaneous normal modes and the resulting spectral line shapes. 36 It has been clearly demonstrated that heterogeneity in the site frequencies needs to be well reproduced in order to obtain agreement with spectra, as shown for ubiquitin by Cho and co-workers.…”

Sequence-Specific Conformational Dynamics of Model Transmembrane Domains Determines Their Membrane Fusogenic Function

“…1,2 At a more subtle level, the helices themselves may bend at hinge regions 3 or exhibit local structural fluctuations involving brief openings of amide hydrogen bonds. 4 Little is currently known about the potential biological significance of these local transmembrane domain (TMD) helix fluctuations. It seems clear, however, that evolutionary fine-tuning can optimize a functional role of helix dynamics if it depends on primary structure.…”