Molecular basis of mRNA transport by a kinesin-1–atypical tropomyosin complex

Abstract

Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances within cells. The mechanochemical cycle of kinesins is well described, but how they establish cargo specificity is not fully understood. Transport of oskar mRNA to the posterior pole of the Drosophila oocyte is mediated by Drosophila kinesin-1, also called kinesin heavy chain (Khc), and a putative cargo adaptor, the atypical tropomyosin, aTm1. How the proteins cooperate in mRNA transport is unknown. Here, we present the high-resolution crystal structure of a Khc-aTm1 complex. The proteins form a tripartite coiled coil comprising two in-register Khc chains and one aTm1 chain, in antiparallel orientation. We show that aTm1 binds to an evolutionarily conserved cargo binding site on Khc, and mutational analysis confirms the importance of this interaction for mRNA transport in vivo. Furthermore, we demonstrate that Khc binds RNA directly and that it does so via its alternative cargo binding domain, which forms a positively charged joint surface with aTm1, as well as through its adjacent auxiliary microtubule binding domain. Finally, we show that aTm1 plays a stabilizing role in the interaction of Khc with RNA, which distinguishes aTm1 from classical motor adaptors.

Stably bound adaptor proteins modulate directionality of RNP transport

“…For RNA transport, adaptor proteins link kinesin-1 and cytoplasmic dynein (hereafter referred to as dynein) to RNA through specific motifs in the RNA (Clark et al, 2007; Dienstbier & Li, 2009; Brendza et al, 2000; Gáspár et al, 2017; Veeranan-Karmegam et al, 2016; Dimitrova-Paternoga et al, 2021). The Drosophila egg chamber exists as a syncytium comprising 15 nurse cells and 1 oocyte, surrounded by a monolayer of follicle cells (Spradling, 1993).…”

“…Once in the oocyte, bicoid and gurken RNAs localize in a dynein-dependent manner to the anterior and anterolateral regions of the oocyte respectively, while oskar localizes in a kinesin-dependent manner to the posterior pole (Brendza et al, 2000; Ferrandon et al, 1994; Thio et al, 2000; Van De Bor et al, 2005; Ghosh et al, 2012). An atypical isoform of Tropomyosin-1, Tm1-I/C ( a Tm1), has been identified as an adaptor protein that binds both kinesin-1 and RNA, and is thought to link kinesin-1 to oskar RNA in developing egg chambers (Gáspár et al, 2017; Veeranan-Karmegam et al, 2016; Dimitrova-Paternoga et al, 2021).…”

Molecular architecture of the autoinhibited kinesin-1 lambda particle

“…The position of the KLC TPR domains is less clear; we favour an interpretation where they are accommodated within the head of the complex, that is larger than would be expected for motor domains alone, and is consistent with previous fluorescence resonance energy transfer data ( 12 ). In which case, the hump would most likely include the AlphaFold2-predicted homodimeric coiled coil of KLC and/or CC4 of KHC that is known to be conformationally plastic, and within which, as we note above, is predicted a proline-induced kink/bulge ( 39, 49 ). Overall, the experimental data indicate that the computational model reflects the gross coiled-coil architecture of the compact conformer of kinesin-1.…”

“…This highlighted a series of heptad repeats indicative of coiled-coil regions in KHC, CC0 to CC4 (Fig. 1A) ( 38 ): CC0 corresponds to the neck coil; CC1-CC3 form the stalk, the latter component of which includes the KLC binding site; and CC4 is known to bind several cargoes/adaptors ( 26, 39–42 ). CC1 and CC2 are separated by a drop in coiled-coil prediction probability ( known as Hinge 2 ) ( 23, 25, 43, 44 ), which has generally been thought to be the point where KHC folds in the autoinhibited state.…”

“…This highlighted a series of heptad repeats indicative of coiled-coil regions in KHC, CC0 to CC4 (Fig. 1A)(38): CC0 corresponds to the neck coil; CC1-CC3 form the stalk, the latter component of which includes the KLC binding site; and CC4 is known to bind several cargoes/adaptors (26,(39)(40)(41)(42).…”