N-terminal splicing extensions of the human gene fine-tune the kinetics of the three full-length myosin IC isoforms.

The gene produces three alternatively spliced isoforms, differing only in their N-terminal regions (NTRs). These isoforms, which exhibit both specific and overlapping nuclear and cytoplasmic functions, have different expression levels and nuclear-cytoplasmic partitioning. To investigate the effect of NTR extensions on the enzymatic behavior of individual isoforms, we overexpressed and purified the three full-length human isoforms from suspension-adapted HEK cells. MYO1C favored the actomyosin closed state (AM), MYO1C populated the actomyosin open state (AM) and AM equally, and MYO1C favored the AM state. Moreover, the full-length constructs isomerized before ADP release, which has not been observed previously in truncated MYO1C constructs. Furthermore, global numerical simulation analysis predicted that MYO1C populated the actomyosin·ADP closed state (AMD) 5-fold more than the actomyosin·ADP open state (AMD) and to a greater degree than MYO1C and MYO1C (4- and 2-fold, respectively). On the basis of a homology model of the 35-amino acid NTR of MYO1C (NTR) docked to the X-ray structure of MYO1C, we predicted that MYO1C NTR residue Arg-21 would engage in a specific interaction with post-relay helix residue Glu-469, which affects the mechanics of the myosin power stroke. In addition, we found that adding the NTR peptide to MYO1C yielded a protein that transiently mimics MYO1C kinetic behavior. By contrast, NTR, which harbors the R21G mutation, was unable to confer MYO1C-like kinetic behavior. Thus, the NTRs affect the specific nucleotide-binding properties of MYO1C isoforms, adding to their kinetic diversity. We propose that this level of fine-tuning within MYO1C broadens its adaptability within cells.