Backbone Dynamics Studies of Mammalian Dynactin CAP-Gly Domain by Solid-State NMR

Abstract

Dynactin is a multisubunit microtubule-associated protein complex functioning in retrograde transport, and binds to microtubules via the CAP-Gly domain of its p150Glued subunit. We have recently reported solid-state NMR investigations of CAP-Gly free and in complex with microtubules.1 Mutations in the CAP-Gly domain of the p150Glued subunit of dynactin are associated with neurological disorders, and little has been known about what gives rise to the pathogenicity associated with these mutations. We have discovered recently that several neurologically related mutants, surprisingly, do not show altered binding to microtubules while their global fold and stability are perturbed with respect to the wild type protein.2 We hypothesize that backbone dynamics of CAP-Gly domain of dynactin may be an important determinant of its interaction with microtubules, and therefore understanding the internal motions in free CAP-Gly and in CAP-Gly/MT complex may provide insights into the regulation mechanisms of CAP-Gly function. Here, we present site-specific measurements of 15N-1H dipolar and 15N CSA lineshapes of CAP-Gly by solid-state NMR spectroscopy at different temperatures, using R1817 and ROCSA recoupling sequences.3,4 The CSA and dipolar order parameters derived from lineshapes by numerical simulations indicate different backbone mobility for the different residues of CAP-Gly; strong correlation between the variation of dipolar order parameters with temperature and the secondary structure is observed.