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

• Published in: The Journal of biological chemistry 2017-10, Vol.292 (43), p.17804-17818
• Main Authors: Zattelman, Lilach, Regev, Ronit, Ušaj, Marko, Reinke, Patrick Y.A., Giese, Sven, Samson, Abraham O., Taft, Manuel H., Manstein, Dietmar J., Henn, Arnon
• Format: Article
• Language: English
• Subjects: Actomyosin - chemistry; Actomyosin - genetics; Actomyosin - metabolism; Adenosine Diphosphate - chemistry; Adenosine Diphosphate - genetics; Adenosine Diphosphate - metabolism; Alternative Splicing; Amino Acid Substitution; Biochemistry; Biokemi; Crystallography, X-Ray; enzyme mechanism; Enzymology; HEK293 Cells; Humans; Isoenzymes; molecular modeling; molecular motor; Mutation, Missense; MYO1C; myosin; Myosin Type I - chemistry; Myosin Type I - genetics; Myosin Type I - metabolism; NMI; pre-steady-state kinetics
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M117.794008

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. 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 the actomyosin·ADP open state (AMDO) and to a greater degree than MYO1CC 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 MYO1CC, 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 MYO1CC 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.