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ACKNOWLEDGMENTS
We thank B. I. Halperin, A. H. Macdonald, N. P. Ong, and P. Kim for
helpful discussions.Funding:Supported by NSF-DMR-1904442
and ONR-N00014-21-1-2592; Gordon and Betty Moore Foundation
EPiQS initiative grant GBMF9469; NSF-MRSEC through the
Princeton Center for Complex Materials NSF-DMR-2011750;
DOE-BES grant DE-FG02-07ER46419; the Princeton Catalysis
Initiative; the Elemental Strategy Initiative, Japan, grant
JPMXP0112101001, JSPS KAKENHI grant JP20H00354, and CREST
(JPMJCR15F3), JST (K.W. and T.T.); and the Army Research
Office through the MURI program (grant W911NF-17-1-0323)
(M.P.Z.). A.Y. acknowledges the hospitality of the Aspen Center for
Physics, which is supported by NSF grant PHY-1607611, and
Trinity College, where his stay was supported by a QuantEmX
grant from ICAM and the Gordon and Betty Moore Foundation
through grant GBMF9616.Author contributions:X.L., G.F.,
C.L.-C., and A.Y. designed the experiment. G.F., X.L., and C.L.-C.
fabricated the sample. X.L., G.F., and C.L.-C. performed the
measurements and analyzed the data. M.P.Z., Z.P., and X.L.
conducted the theoretical analysis. K.W. and T.T. provided hBN
crystals. X.L., G.F., C.L.-C., A.Y., and M.P.Z. wrote the manuscript
with input from all authors.Competing interests:The authors
declare no competing interests.Data and materials availability:The
data from this study are available at the Harvard Dataverse ( 48 ).

SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abm3770
Materials and Methods
Supplementary Text
Figs. S1 to S7
References ( 49 – 59 )

13 September 2021; accepted 17 November 2021
Published online 2 December 2021
10.1126/science.abm3770

MOLECULAR MOTORS

Structural and functional insight into regulation of

kinesin-1 by microtubule-associated protein MAP7

Luke S. Ferro^1 †, Qianglin Fang^1 *†‡, Lisa Eshun-Wilson^1 †, Jonathan Fernandes^2 †, Amanda Jack^3 ,

Daniel P. Farrell^4 , Mert Golcuk^5 , Teun Huijben^6 , Katelyn Costa^7 , Mert Gur^5 , Frank DiMaio^4 ,

Eva Nogales1,3,8,9*, Ahmet Yildiz1,3,9,10*

Microtubule (MT)–associated protein 7 (MAP7) is a required cofactor for kinesin-1–driven transport

of intracellular cargoes. Using cryo–electron microscopy and single–molecule imaging, we investigated

how MAP7 binds MTs and facilitates kinesin-1 motility. The MT-binding domain (MTBD) of MAP7 bound

MTs as an extendedahelix between the protofilament ridge and the site of lateral contact. Unexpectedly,

the MTBD partially overlapped with the binding site of kinesin-1 and inhibited its motility. However, by tethering

kinesin-1 to the MT, the projection domain of MAP7 prevented dissociation of the motor and facilitated its

binding to available neighboring sites. The inhibitory effect of the MTBD dominated as MTs became saturated

with MAP7. Our results reveal biphasic regulation of kinesin-1 by MAP7 in the context of their competitive

binding to MTs.

K

inesin and dynein are molecular motors

that deliver intracellular cargos by walk-

ing along microtubules (MTs) ( 1 , 2 ).

Intracellular cargos are differentially

regulated by structural MT-associated

proteins (MAPs) that decorate the MT surface

( 1 ). Distinct cellular localizations of MAPs cor-

relate with their regulatory roles in intracellu-

lar traffic ( 3 ). Overexpression of tau disrupts

kinesin-1 (hereafter kinesin)–driven transport

of synaptic vesicles in axons ( 4 , 5 ), whereas the

knockdown of tau rescues defects in axonal

transport in Alzheimer’sdiseasemodels( 6 ).

Unlike tau, MAP7 is a required cofactor for

kinesin-driven transport in cells ( 7 – 9 ). The MAP7

projection domain binds to kinesin’s coiled-coil

stalk in vitro ( 9 , 10 ), recruits kinesin to MTs,

and activates its motility ( 9 , 11 ). Transient in-

teractions with the MAP7 projection domain

may enable kinesin to hop from one MAP to

another, increasing its apparent run length by

disfavoring detachment from the MT ( 9 ).

To understand how MAP7 regulates kinesin,

we determined the cryo–electron microscopy

(cryo-EM) structure of MTs decorated with

full-length (FL) MAP7 (Fig. 1, A and B; fig. S1A;

tables S1 and S2; and movie S1). The recon-

struction revealed a 53-residue-longahelix

that runs parallel to the MT axis about the

length of a tubulin dimer (Fig. 1, A to C, and

fig. S1B). Unlike MAP2, MAP4, and tau, which

bind along the outer ridges of the protofila-

ments ( 12 – 14 ), MAP7 runs halfway between

the outer ridge and the site of lateral contact.

The costructure of MAP7’s MT binding do-

main (MTBD; residues 60 to 170) and FL tau

on the MT (fig. S2) illustrated their distinct

MT footprints and confirmed that the helical

segment corresponds to the MTBD of MAP7.

We identified a single MAP7 sequence register

that corresponds to a well-conserved segment

of the MTBD (residues 87 to 139; fig. S3, A to C)

through Rosetta modeling ( 15 ) and validated

this registry by determining the structure of

MTs decorated with a shorter MAP7 construct

(residues 83 to 134; fig. S3D). Because MAP7

MTBD could potentially form a helix longer

than the length of a tubulin heterodimer (fig.

S2A),wecannotexcludethepossibilityofa

larger footprint of MAP7 on the MT ( 13 ) (fig.

S4; see materials and methods).

Thea-helical density for MAP7 is not uni-

form (Fig. 1B). Segment I (residues 113 to 139)—

the best-resolved region (Fig. 1B)—interacts

extensively with tubulin (Fig. 1D); Q113 and

E117 of MAP7 are within hydrogen-bonding

distance of N197 and S155 ofb-tubulin, re-

spectively; R114 and K127 of MAP7 engage in

electrostatic interactions with E159 and D414

ofb-tubulin, respectively. Y108 ofb-tubulin in-

serts into a hydrophobic pocket formed by

R120, R121, and V124 of MAP7. We also iden-

tified potential hydrogen bonds between R128,

R131, and K136 of MAP7 and the mainchain

oxygens of E411 and G410 ofb-tubulin and V159

ofa-tubulin, respectively. Segment III (residues

87 to 99) interacts witha-tubulin. Segment II

(residues 100 to 113) faces a cavity at the intra-

tubulin dimer and has the weakest density in

our map (Fig. 1B). All-atom molecular dynam-

ics (MD) simulations verified these pairwise

326 21 JANUARY 2022•VOL 375 ISSUE 6578 science.orgSCIENCE

(^1) Department of Molecular and Cell Biology, University of
California, Berkeley, CA, USA.^2 Department of Chemistry,
University of California, Berkeley, CA, USA.^3 Biophysics Graduate
Group, University of California, Berkeley, CA, USA.^4 Department
of Biochemistry, University of Washington, Seattle, WA, USA.
(^5) Department of Mechanical Engineering, Istanbul Technical
University, Istanbul, Turkey.^6 Department of Imaging Physics,
Delft University of Technology, Delft, Netherlands.^7 Press West
Illustrations, Boston, MA, USA.^8 Howard Hughes Medical
Institute, University of California, Berkeley, CA, USA.^9 Molecular
Biophysics and Integrative Bioimaging Division, Lawrence
Berkeley National Laboratory, Berkeley, CA, USA.^10 Physics
Department, University of California, Berkeley, CA, USA.
*Corresponding authors. [email protected] (A.Y.); enogales@
lbl.gov (E.N.); [email protected] (Q.F.)
†These authors contributed equally to this work.
‡Present address: School of Public Health, Sun Yat-sen University,
Shenzhen, China.
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