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To position the nucleus for neuronal migration, Nesprin-2 interacts with the motors kinesin-1 and dynein, which are recruited by the adapter Bicaudal D2 (BicD2), but the molecular details of these interactions are elusive. Here, structural models of minimal Nesprin-2\/BicD2 complexes with 1:2 and 2:2 stoichiometry were predicted using AlphaFold and experimentally validated by mutagenesis, binding assays and single-molecule biophysical studies. The core of the binding site is formed by spectrin repeats of Nesprin-2, which form an alpha-helical bundle with BicD2 that is structurally distinct from the Rab6\/BicD2 and Nup358\/BicD2 complexes. Such structural differences could fine tune motility of associated dynein and kinesin-1 motors for these transport pathways. Furthermore, the Nesprin-2 fragment interacts with full-length BicD2 and activates dynein\/dynactin\/BicD2 complexes for processive motility, suggesting that no additional components are required to reconstitute this transport pathway. Interestingly, either one or two Nesprin-2 molecules can bind to a BicD2 dimer and activate BicD2\/dynein\/dynactin complexes for processive motion, resulting in similar speed and run lengths. The BicD2\/dynein-binding site is spatially close but does not overlap with the kinesin-1 recruitment site, thus both motors may interact with Nesprin-2 simultaneously. Several mutations of Nesprin-1 and 2 that cause Emery-Dreifuss muscular dystrophy are found in the motor-recruiting domain and may alter interactions with kinesin-1 and BicD2\/dynein, consistent with the abnormally positioned nuclei found in patients with this disease.<\/jats:p>","DOI":"10.1101\/2025.05.18.654709","type":"posted-content","created":{"date-parts":[[2025,5,23]],"date-time":"2025-05-23T09:50:15Z","timestamp":1747993815000},"source":"Crossref","is-referenced-by-count":0,"title":["Cargo recognition of Nesprin-2 by the dynein adapter Bicaudal D2 for a nuclear positioning pathway that is important for brain development"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-5545-5470","authenticated-orcid":false,"given":"Estrella D","family":"Rodriguez Castro","sequence":"first","affiliation":[{"name":"Department of Chemistry, Binghamton University, PO Box 6000, Binghamton, NY 13902, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3071-8526","authenticated-orcid":false,"given":"Sivasankar","family":"Putta","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Binghamton University, PO Box 6000, Binghamton, NY 13902, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2164-3323","authenticated-orcid":false,"given":"M. Yusuf","family":"Ali","sequence":"additional","affiliation":[{"name":"Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jose M","family":"Garcia Martin","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Binghamton University, PO Box 6000, Binghamton, NY 13902, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoxin","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Binghamton University, PO Box 6000, Binghamton, NY 13902, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samantha","family":"Sylvain","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Binghamton University, PO Box 6000, Binghamton, NY 13902, 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