Delineating elastic properties of kinesin linker and their sensitivity to point mutations

Abstract

We analyze free energy estimators from simulation trials mimicking single-molecule pulling experiments on a neck linker of a kinesin motor. For that purpose, we have performed a version of steered molecular dynamics (SMD) calculations. the sample trajectories have been analyzed to derive distribution of work done on the system. In order to induce stretching of the linker, we have applied a constant pulling force to the molecule and allowed for a subsequent relaxation of its structure. the use of fluctuation relations (FR) relevant to non-equilibrium systems subject to thermal fluctuations allows us to assess the difference in free energy between stretched and relaxed conformations. To further understand effects of potential mutations on elastic properties of the linker, we have performed similar in silico studies on a structure formed of a polyalanine sequence (Ala-only) and on three other structures, created by substituting selected types of amino acid residues in the linker's sequence with alanine (Ala) ones. the results of SMD simulations indicate a crucial role played by the Asparagine (Asn) and Lysine (Lys) residues in controlling stretching and relaxation properties of the linker domain of the motor.The motor proteins' ability to generate movement creates a situation, where it is possible to treat different parts of one molecule as linked but functionally separate objects, that possess an ability to move at different times and speeds 1 . That makes the field of motor proteins a desirable testing ground for application of various theoretical models, that aim to filter the inherent complexity of biological systems 2-5 . Among the motors, members of the kinesin protein superfamily are consistently used throughout the years in research focusing on molecular motors' mechanical properties, both in vitro 2 and in silico 2,4-6 .A conformational change of a region within kinesin has been shown to be associated with its movement along microtubule 7 . This region has been labeled as neck linker. The label refers to a concise (less than 20 amino acids length) amino acid sequence in a single kinesin head that acts as a bridge between α-6 helix in coiled-coil dimerization domain and α-7 helix in the core motor domain, respectively 8 . While its exact length as well as the placement within sequence of its N-and C-termini are not set in stone 9 , some of its residue patterns are present across all kinesin families, while neck linker sequences within a single family are very similar 8 . All this suggest that parts of neck linker region have been conserved, which makes neck linker a non-random, specific sequence. An on-going accumulation of experimental data evidence suggests that a transition of the neck linker from a disordered (random coil) state to an ordered (β-sheet) conformation is a key factor in determining a mechanism of force-generation that is a crucial element of molecular motors' ability to move along microtubules 7,8 . Attempts at substituting neck linker with a peptide of a different sequence, as well as exte...