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.
Myosin-1C is a single-headed, short-tailed member of the myosin class I subfamily that supports a variety of actin-based functions in the cytosol and nucleus. In vertebrates, alternative splicing of the MYO1C gene leads to the production of three isoforms, myosin-1C0, myosin-1C16 and myosin-1C35, that carry N-terminal extensions of different length. However, it is not clear how these extensions affect the chemomechanical coupling of human myosin-1C isoforms. Here, we report on the motor activity of the different myosin-1C isoforms measuring the unloaded velocities of constructs lacking the C-terminal lipid binding domain on nitrocellulose-coated glass surfaces and full-length constructs on reconstituted, supported lipid bilayers. The higher yields of purified protein obtained with constructs lacking the lipid binding domain allowed a detailed characterization of the individual kinetic steps of human myosin-1C isoforms in their productive interaction with nucleotides and filamentous actin. Isoform-specific differences include 18-fold changes in the maximum power output per myosin-1C motor and 4-fold changes in the velocity and the resistive force at which maximum power output occurs. Our results support a model in which the isoform-specific N-terminal extensions affect chemomechanical coupling by combined steric and allosteric effects, thereby reducing both the length of the working stroke and the rate of ADP release in the absence of external loads by a factor of two for myosin-1C35. As the large change in maximum power output shows, the functional differences between the isoforms are further amplified by the presence of external loads.
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