N-terminal splicing extensions of the human MYO1C gene fine-tune the kinetics of the three full-length myosin IC isoforms [Molecular Biophysics]

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 nucleo−cytoplasmic partitioning. To investigate the effect of NTR extensions on the enzymatic behavior of individual isoforms, we over−expressed and purified the three full−length human isoforms from suspension−adapted HEK cells. MYO1CC favored the actomyosin closed state (AMC), MYO1C16 populated the actomyosin open state (AMO) and AMC equally, and MYO1C35 favored the AMO state. Moreover, the full−length constructs isomerized before ADP release, which has not been observed previously in truncated MYO1CC constructs. Furthermore, global numerical simulation analysis predicted that MYO1C35 populated the actomyosin·ADP closed state (AMDC) 5−fold more than actomyosin·ADP open state (AMDO) and to a greater degree than MYO1CC and MYO1C16 (4−fold 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 MYO1CC, we predicted that a MYO1C35 NTR residue Arg−21 engages in a specific interaction with post−relay helix residue Glu−469 that affects the mechanics of the myosin power stroke. In addition, we found that adding the NTR35 peptide to MYO1CC yielded a protein that transiently mimics MYO1C35 kinetic behavior. By contrast, NTR35 harboring the R21G mutation was unable to confer MYO1C35−like kinetic behavior. Therefore, 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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