The MYO1C 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^C favored the actomyosin closed state (AM^C), MYO1C¹⁶ populated the actomyosin open state (AM^O) and AM^C equally, and MYO1C³⁵ favored the AM^O state. Moreover, the full-length constructs isomerized before ADP release, which has not been observed previously in truncated MYO1C^C constructs. Furthermore, global numerical simulation analysis predicted that MYO1C³⁵ populated the actomyosin·ADP closed state (AMD^C) 5-fold more than the actomyosin·ADP open state (AMD^O) and to a greater degree than MYO1C^C and MYO1C¹⁶ (4- and 2-fold, respectively). On the basis of a homology model of the 35-amino acid NTR of MYO1C³⁵ (NTR³⁵) docked to the X-ray structure of MYO1C^C, we predicted that MYO1C³⁵ 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 NTR³⁵ peptide to MYO1C^C yielded a protein that transiently mimics MYO1C³⁵ kinetic behavior. By contrast, NTR³⁵, which harbors the R21G mutation, was unable to confer MYO1C³⁵-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.

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