NMR characterisation of the conformational fluctuations of the human lymphocyte function associated antigen-1 i domain

[nmrlearner]

NMR characterisation of the conformational fluctuations of the human lymphocyte function associated antigen-1 i domain

Abstract

Lymphocyte function-associated antigen-1 (LFA-1) is an integrin protein that transmits information across the plasma membrane through the so-called ‘inside-out’ and ‘outside-in’ signalling mechanism. To investigate this mechanism, we carried out an NMR analysis of the dynamics of the LFA-1 I-domain, which has enabled us to characterise the motions of this domain on a broad range of timescales. We studied first the internal motions on the nanosecond timescale by spin relaxation measurements and model-free analysis. We then extended this analysis to the millisecond timescale motions by measuring 15N-1H residual dipolar couplings (RDCs) of the backbone amide groups. We analysed these results in the context of the three major conformational states of the I-domain using their corresponding X-ray crystallographic structures. Our results highlight the importance of the low-frequency motions of the LFA-1 I-domain in the inactive apo state. We found in particular that ?-helix 7 is in a position in the apo closed state that cannot be fully described by any of the existing X-ray structures, as it appears to be in dynamic exchange between different conformations. This type of motion seems to represent an inherent property of the LFA-1 I-domain and might be relevant for controlling the access to the allosteric binding pocket, as well as for the downward displacement of ?-helix 7 that is required for the activation of LFA-1.




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