Neck linker length determines the degree of processivity in kinesin-1 and kinesin-2 motors.

Abstract

Defining the mechanical and biochemical determinates of kinesin processivity is important for understanding how diverse kinesins are tuned for specific cellular functions. Because transmission of mechanical forces through the 14-18 amino acid neck linker domain underlies coordinated stepping, we investigated the role of neck linker length, charge, and structure in kinesin-1 and kinesin-2 motor behavior. For optimum comparison with kinesin-1, the KIF3A head and neck linker of kinesin-2 were fused to the kinesin-1 neck coil and rod. Extending the 14-residue kinesin-1 neck linker reduced processivity, and shortening the 17-residue kinesin-2 neck linker enhanced processivity. When a proline in the kinesin-2 neck linker was replaced, kinesin-1 and kinesin-2 run lengths scaled identically with neck linker length, despite moving at different speeds. In low-ionic-strength buffer, charge had a dominant effect on motor processivity, which resolves ongoing controversy regarding the effect of neck linker length on kinesin processivity. From stochastic simulations, the results are best explained by neck linker extension slowing strain-dependent detachment of the rear head along with diminishing strain-dependent inhibition of ATP binding. These results help delineate how interhead strain maximizes stepping and suggest that less processive kinesins are tuned to coordinate with other motors differently than the maximally processive kinesin-1.

Full text

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Current Biology 20, 939–943, May 25, 2010 ª2010 Elsevier Ltd All rights reservedDOI 10.1016/j.cub.2010.03.065
Report
Neck Linker Length Determines
the Degree of Processivity
in Kinesin-1 and Kinesin-2 Motors
Shankar Shastry1and William O. Hancock1,*
1Department of Bioengineering, The Pennsylvania State
University, 205 Hallowell Building, University Park, PA 16802,
USA
Summary
Defining the mechanical and biochemical determinates of
kinesin processivity is important for understanding how
diverse kinesins are tuned for specific cellular functions.
Because transmission of mechanical forces through the
14–18 amino acid neck linker domain underlies coordinated
stepping [1–6],we investigated theroleofneck linker length,
charge, and structure in kinesin-1 and kinesin-2 motor
behavior. For optimum comparison with kinesin-1, the
KIF3A head and neck linker of kinesin-2 were fused to the
kinesin-1 neck coil and rod. Extending the 14-residue kine-
sin-1 neck linker reduced processivity, and shortening the
17-residue kinesin-2 neck linker enhanced processivity.
When a proline in the kinesin-2 neck linker was replaced,
kinesin-1 and kinesin-2 run lengths scaled identically with
neck linker length, despite moving at different speeds. In
low-ionic-strength buffer, charge had a dominant effect on
motor processivity, which resolves ongoing controversy
regarding the effect of neck linker length on kinesin proces-
sivity [3, 5–7]. From stochastic simulations, the results are
best explained by neck linker extension slowing strain-
dependent detachment of the rear head along with diminish-
ing strain-dependent inhibition of ATP binding. These
results help delineate how interhead strain maximizes step-
ping and suggest that less processive kinesins are tuned to
coordinate with other motors differently than the maximally
processive kinesin-1.
Results and Discussion
Extending the Kinesin-1 Neck Linker Decreases
Processivity
To investigate the influence of neck linker length on kinesin-1
processivity, we visualized homodimeric Drosophila con-
ventional kinesin, truncated at residue 559 and fused to
a C-terminal GFP, moving along immobilized bovine brain
microtubules by single-molecule total internal reflection fluo-
rescence (TIRF) microscopy. To minimize any electrostatic
tethering between the motor and microtubule that can compli-
cate the interpretation of mechanical processivity, we per-
formed experiments in 80 mM PIPES buffer. The control Kin1
motor moved at 990 6 130 nm/s (mean 6 standard deviation),
with a mean run length of 2.1 6 0.1 mm (mean 6 standard error
of fit) (Figure 1A). When the neck linker domain was extended
by three residues (Kin1+DAL, corresponding to the last three
residues of the kinesin-2 neck linker), the run length dropped
by a factor of five to 0.39 6 0.02 mm and the speed dropped
to 600 6 89 nm/s. Interestingly, inserting only one amino
acid (Kin1+L) diminished the run length to 0.89 6 0.06 mm and
the speed to 580 6 95 nm/s (Figure 1B). Finally, shortening
the neck linker by one residue (Kin1DT) abolished processivity
(see Supplemental Results and Discussion and Figure S1
available online). These results confirm and extend previous
work with full-length kinesin attached to beads [6] and demon-
strate that kinesin-1 processivity is significantly reduced when
the neck linker domain is extended by even one amino acid.
Shortening the Kinesin-2 Neck Linker Enhances
Processivity
The intraflagellar transport motor kinesin-2 has a 17 amino
acid neck linker, three residues longer than kinesin-1, and in
previous work we showed that full-length kinesin-2 is 4-fold
less processive than kinesin-1 [6]. If the reduced processivity
of kinesin-2 results from diminished coordination between
the heads that results from its longer neck linker domain,
then a simple prediction is that shortening the kinesin-2 neck
linker domain should enhance processivity. To test this, we
made a motor consisting of the head and neck linker of the
mouse KIF3A subunit of kinesin-2 (ending in the last residue
of the neck linker domain, Leu359) fused to the neck coil and
proximal rod of Drosophila kinesin-1 (starting at the first
residue of the neck coil domain, Ala345) (Figure 1C). This kine-
sin-2 construct was used so that any differences in processiv-
ity can be attributed solely to the head and neck linker regions,
and not to differences such as charge or mechanical integrity
of the coiled coil. Whereas constructs containing the kinesin-2
coiled coil were only functional when they were baculovirus-
expressed, these kinesin-1/kinesin-2 chimeras were func-
tional when bacterially expressed and had similar properties
to a baculovirus-expressed KIF3A homodimer investigated
previously [6]. These GFP-tagged chimeric constructs are
referred to as Kin2 throughout this paper.
The mean run length and speed of control Kin2 were 0.71 6
0.03 mm and 480 6 98 nm/s, respectively. To test whether
shortening the neck linker enhances processivity, we made
stepwise deletions of one, two, and three amino acids in the
last three residues (DAL) in the kinesin-2 neck linker to create
Kin2DA, Kin2DDA, and Kin2DDAL. Single-molecule run lengths
and velocities were measured in a manner identical to Kin1.
Deleting one residue (Kin2DA) increased the run length to
1.26 mm and had no effect on the velocity (484 nm/s), support-
ing the hypothesis. However, deleting two residues (Kin2DDA)
decreased the mean run length to 0.59 mm, which is less
than the control Kin2 motor. Deleting all three residues
(Kin2DDAL) resulted in no observable processive runs in the
single-molecule assay (Figure 2C), though the motors were
functional in the multimotor gliding assay (Supplemental
Results and Discussion and Table S2). Hence, although the
results qualitatively agree with the hypothesis—shortening
the kinesin-2 neck linker enhances processivity—there was
not quantitative agreement between the kinesin-1 and kine-
sin-2 results.
Both KIF3A and KIF3B contain a proline residue at position
13 of the neck linker domain (Figure 1C), and in the only kine-
sin-2 crystal structure containing the entire neck linker domain
(humanKIF3B;ProteinDataBankIDcode3B6U),thisprolineis
*Correspondence: wohbio@engr.psu.edu

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in the kinked cis conformation. We used molecular dynamics
simulations to compare the predicted force-extension profiles
ofkinesin-1andkinesin-2necklinkersandfoundthat,whereas
the kinesin-1 neck linker was well fitby amodel of a14-residue
worm-like chain, the 17-residue kinesin-2 neck linker was
shorter than predicted by a worm-like chain model, except at
high forces where the proline was forced into the straight trans
conformation [8] (Figure 2A). When the proline at position 13
was changed to an alanine and the last three residues of the
kinesin-2 neck linker were deleted, the modeled force-exten-
sion curve matched that of kinesin-1 (Figure 2B).
Kin1 and Kin2 Motor Properties Scale Similarly with Neck
Linker Length
Motivated by these molecular dynamics simulations, we
substituted the proline at position 13 of the Kin2 neck linker
with alanine (Kin2PA) and found that the mean run length fell
from 0.71 mm to 0.39 mm, consistent with this substitution
extending the effective neck linker length. Interestingly, the
processivity of this Kin2PAconstruct closely matched that of
the equivalent kinesin-1 construct, Kin1+DAL(0.39 mm) (Fig-
ure 2D; Table S2). More importantly, when the neck linker of
this proline-substituted construct was shortened by three
Figure 1. Kin1 Run Lengths and Design of Kin1
and Kin2 Constructs
(A) Run length of control Kin1 from total internal
reflection fluorescence assay. Data were fit to
a single exponential.
(B) Run lengths of different Kin1 constructs as
a function of their neck linker (NL) length. Error
bars represent the standard error from exponen-
tial fits.
(C) Amino acid sequence of the kinesin-1 (KHC)
and kinesin-2 (KIF3A) neck linkers with the adja-
cent a6 (last helix in the head domain) and a7
(neck coiled-coil domain). The Kin1 construct
includes the entire DmKHC sequence up to
residue 559 (in the break between coil 1 and coil
2 of the rod domain [19]) followed by a C-terminal
GFP and His6tag. The Kin2 construct includes
the KIF3A head and 17-residue neck linker
domain ending at Leu359 (red sequence) fused
to the DmKHC neck coil and rod, starting at
Ala345, the first residue in the neck coil domain
(dark blue sequence). The cartoon shows the
structures of the Kin1 and Kin2 constructs. The
neck linker sequences for all constructs used
are given in Table S1.
Figure 2. Kin1 and Kin2 Run Lengths Scale with
Neck Linker Length
(A) Force-extension curves of kinesin-1 and
kinesin-2 neck linkers from molecular dynamics
simulations. Solid lines are predictions of a
worm-like chain model for 14- and 17-residue
polypeptides showing good fit for kinesin-1 (14
residues) and poor fit for kinesin-2 (17 residues).
Data are replotted from [8].
(B) Predicted force-extension curve when the
kinked proline in kinesin-2 is replaced with an
alanineandthelast threeamino acidsaredeleted
(Kin2PA_DDAL), compared to kinesin-1.
(C) Comparison of run lengths for Kin2 and Kin1
constructs with identical neck linker lengths
(number of amino acids).
(D) Run lengths following substitution of the cis
proline in the Kin2 neck linker with alanine
(Kin2PA). Kin2PAconstructs containing 14-, 15-,
and 17-residue neck linkers are Kin2PA_DDAL,
Kin2PA_DDA, and Kin2PA, respectively. The curve
for control Kin2 motors is shifted 1.5 amino acids
to the left to account for the cis proline. All run
length and velocity values are given in Table S2.
Current Biology Vol 20 No 10
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residues to match the length of the kinesin-1 neck linker, the
mean run length of this Kin2PA_DDALconstruct rose to 1.8 mm,
more than twice the native Kin2 processivity and very nearly
matching the 2.1 mm run length of wild-type Kin1 (Figure 2D).
To further examine this correlation, we then made an interme-
diate length construct, Kin2PA_DDA, containing a 15-residue
neck linker domain and found that its run length closely
matched the kinesin-1 construct having a 15-residue neck
linker, Kin1+L. The striking result here is that when the kine-
sin-2 neck linker is straightened by removing the cis proline,
the run lengths of kinesin-1 and kinesin-2 motors match and
scale similarly with neck linker length even though motor
velocities remain distinct. As an example, the run lengths of
Kin1 and Kin2PA_DDALare nearly identical, although their motor
velocities differ by nearly a factor of two (990 6 130 nm/s
versus 508 6 71 nm/s).
To extend this correlation of processivity with neck linker
length,itispossibletoestimatethedegreetowhichtheproline
in the cis conformation shortens the kinesin-2 neck linker.
Shortening the Kin2 neck linker by one residue increases
processivity,whereasshorteningitbytworesiduesdiminishes
processivity and shortening it by three abolishes processivity
altogether (Figure 2C). This is consistent with the maximum
predicted run length lying between a deletion of one and two
residues.WhentheKin2curveisshiftedtotheleftby1.5amino
acids, all three curves fall on top of one another (Figure 2D),
suggesting that the kinked proline shortens the neck linker
domain by the equivalent of 1.5 amino acids (w0.5 nm).
Adding Positive Charge in the Neck Linker Enhances
Processivity
Although it is clear that neck linker length controls processiv-
ity, the degree to which charged residues in the neck linker
also affect processivity is not clear. Positively charged resi-
dues in the neck coil domain and in the core head domain
have been shown to enhance processivity through favorable
electrostatic interactions with the microtubule [9, 10]. Under-
standing the dependence of kinesin processivity on neck
linkerchargeandbufferionicstrength isimportantforproperly
interpreting the present data and for resolving disparate
results in the literature. To test the extent to which the reduced
run length of Kin1+DALis due to unfavorable electrostatic inter-
actions caused by the negative charge of the insert (21 at
physiological pH), we instead inserted a neutral three-residue
insert, AAL. Kin1+AALhad a similar run length (0.45 mm; Fig-
ure 3A), confirming that the DAL insert reduces processivity
by lengthening the neck linker and not by introducing negative
charge. In contrast, extending the neck linker with a positively
charged insert, KAL (Kin1+KAL), resulted in a run length of
1.27 mm, which is more than 2-fold greater than Kin1+DALor
Kin1+AALbut is still considerably less processive than Kin1
(2.1 mm) (Figure 3A). Hence, even in 80 mM PIPES buffer, posi-
tively charged residues in the kinesin neck linker domain
enhance processivity. However, in this case, the reduction in
processivity due to lengthening the neck linker still clearly
dominates over any electrostatic effects from the positively
charged lysine.
If electrostatic interactions are playing a role in kinesin proc-
essivity, then the effect should be magnified in low-ionic-
strength buffers where charge shielding is minimized. To test
the effect of ionic strength on kinesin processivity, we mea-
sured run lengths of Kin1 and Kin1+KALin 12 mM PIPES buffer
andcomparedthevaluestorunlengthsin80mMPIPESbuffer.
In 12 mM PIPES, the Kin1 run length doubled to 4.2 mm and the
Kin1+KALrun length increased 4-fold to 4.6 mm, such that the
Kin1andKin1+KALrunlengthswerenearlyidentical(Figure3B).
Hence, in low-ionic-strength buffers, the reduction in proces-
sivity resulting from extending the neck linker was almost
perfectly matched by the enhancement in processivity result-
ing from the positively charged lysine in the insert.
This finding that charge introduced in the neck linker plays
a dominating role at low ionic strength helps to resolve the
disparity between the present data and the results of Yildiz
et al. [5], who found that inserts doubling the neck linker length
had no significant effect on processivity. In that work, every
insert in the neck linker contained an additional two lysines
and a glycine; the authors argued that these positive charges
compensated for moving the normal positive charge in the
neck coil domain farther from the microtubule. Based on our
results, the enhanced electrostatic interactions from these
two lysines, which will be amplified in the 12 mM PIPES
buffer used in that study, overwhelmed any reduction in proc-
essivity resulting from extending the neck linker domains.
The simplest explanation is that these positively charged
residues enhance processivity by interacting with the nega-
tively charged C terminus of tubulin [9–11], although other
mechanisms cannot be ruled out. We argue that for under-
standing the chemomechanical coordination between the
two head domains that underlies kinesin processivity, these
electrostatic effects should be minimized by using higher-
ionic-strength buffers and minimizing positive charge in any
sequence inserts.
Figure 3. Neck Linker Positive Charge and Low Ionic Strength Enhance
Processivity
(A) Kin1 run length as a function of the charge of the neck linker insert,
showing that althoughnegativecharge does not diminish processivity, add-
ing positive charge does enhance processivity. Experiments were carried
out in 80 mM PIPES buffer.
(B) Effect of buffer ionic strength on control Kin1 and Kin1+KALrun lengths,
showing that in 12 mM PIPES buffer, the diminished processivity due to the
longer neck linker domain is compensated for by enhanced electrostatic
interactions due to the added positive charge in the neck linker domain.
Neck Linker Length Determines Processivity
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How Does Extending the Neck Linker Alter the Kinesin
Chemomechanical Cycle?
To understand the mechanism by which extending the kinesin
neck linker reduces processivity by a factor of five while
reducing velocity less than 2-fold, we carried out stochastic
simulations of the kinesin-1 kinetic cycle (Figure 4A) to identify
whether modifications of individual model parameters are
sufficient to account for the experimental results. Kinesin
processivity is described by two mechanisms, front-head
and rear-head gating [2, 4, 6]. Front-head gating holds that
when both heads are bound (state 1), ATP binding to the
leading head is inhibited by rearward strain, ensuring that
the trailing head detaches before ATP binds (state 2). Rear-
head gating holds that detachment from a one-head-bound
state (state 5) is slow and forward-directed strain from the
second head is necessary to detach the rear head (state 1 to
2 or state 3 to 4) at a rate consistent with the overall cycle
time [4, 12].
For wild-type kinesin-1, where front-head gating is thought
to block state 3 [2], processivity is determined by the relative
rates of unbinding of the attached head (state 5 to state 6)
versus rebinding of the tethered head (state 5 to state 1). To
test whether changes in kattach alone can account for the
experimental results, we ran simulations at a range of kattach
values while holding all other parameters constant. Depending
on the specific parameters used, the experimental results
could be accounted for by positing that extending the neck
linker reduces kattach(Figure S4; Table S3). However, because
attachment involves tethered diffusion of the unbound head,
constrained by the entropic elasticity of the neck linker [6, 8,
13], extending the neck linker would be expected if anything
to increase kattachrather than decrease it. Nonetheless, this
mechanism remains a formal possibility.
The next mechanism tested was the possibility that extend-
ing the kinesin-1 neck linker disrupts front-head gating by
increasing the ATP binding rate in the two-head-bound state
(state 1 to state 3 transition). To test whether a change in
ATP binding alone is sufficient to account for the data, we
varied kon_ATP_2Hfrom 0.02 mM21s21(consistent with experi-
mental estimates in the strained state [2]) to 2 mM21s21(the
unstrained rate [2, 14]). A steep fall in run length was indeed
observed, but velocity was unchanged (Figure S4). Hence,
although this proposed mechanism can account for the effect
of neck linker extension on run length, it cannot account for
changes in velocity.
The third mechanism tested was the possibility that extend-
ing the neck linker domain slows strain-induced detachment
of the trailing head. This strain-dependent detachment not
only underlies rear-head gating, it also underlies front-head
gating—detachment of the trailing head in state 1 must be
very fast to prevent ATP binding to the front head and possible
detachment from state 3. Depending on the rate constants
chosen, decreasing the strain-induced detachment of the
rear head (an expected outcome of lengthening the neck
Figure 4. Modeling the Kinesin Chemomechanical Cycle
(A) Model for the kinesin chemomechanical cycle used to interpret the neck linker extension results. This framework is similar to a previous model [6], with
the difference that motor unbinding from state 3 is combined into one rate constant (kunbind_2H) for simplicity. Kinetic parameters are discussed in Supple-
mental Results and Discussion and listed in Table S3.
(B) Experimental Kin1 run length and velocity results, plotted as a function of neck linker length.
(C) Modeled run length and velocity from stochastic simulations of the model presented in (A), using rate constant parameters given in Table S3. In these
simulations, kon_ATP_2Hwas set to 0.2 mM21s21(10-fold above the best estimate from the literature [2]) and both kdetach_4and kdetach_Twere varied from
2000 s21down to 200 s21to model the effect of reduced strain on the trailing head due to extending the neck linker domain. Hence, for the model to account
for the experimental results, extending the neck linker needs to alter two strain-dependent mechanisms—detachment of the trailing head and ATP binding
to the leading head. Additional simulation results are given in Figure S4.
Current Biology Vol 20 No 10
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linker) does result in a steep fall in run length and a moderate
decrease in velocity (Figure 4C). However, this result is depen-
dent on setting kon_ATP_2Hto a value 10-fold faster than the
experimentally estimated rate [2], setting kdetachto be very
fast (2000 s21), and setting the unbinding rate from the two-
head-bound state 3 to be 10-fold faster than the one-head-
bound unbinding rate (state 5) (Table S3). Hence, changing
either the ATP binding rate alone or the strain-dependent
detachment rate of the trailing head alone fails to account
for the experimental results. Instead, the neck linker exten-
sion results are best accounted for by both a slowing of
the strain-dependent detachment rate kdetachand an increase
in the strain-suppressed two-headed ATP binding rate
kon_ATP_2H.
The striking finding of this study is that virtually all of the
difference in processivity between kinesin-1 and kinesin-2
motors results from differences in the length of the neck linker
domain,andnotfrominherentdifferencesinkineticratesinthe
heads. Hence, when Kin1 and Kin2 neck linkers are identical
lengths (following proline substitution), theirrun lengths match
despite the fact that motor velocities differ by nearly a factor
of two. The results can be accounted for by proposing that
extending the neck linker both decreases strain-induced
detachment of the trailing head (kdetach) and relieves the
strain-inhibited binding of ATP to the leading head in the two-
head-bound state. Because internal strain between the heads
in kinesin-2 motors is less than in kinesin-1, it is expected
that kinesin-2 responds differently to external strain such
as during bidirectional transport of cargo or when many
motors are cooperatively transporting cargo. Sequence pre-
dictions indicate that motors in different kinesin families have
differentnecklinkerlengths[8],andtheyareknowntopossess
different degrees of processivity and work in diverse multi-
motor arrangements; hence, this correlation of neck linker
length with their cellular task may extend across the kinesin
superfamily.
Experimental Procedures
Motor Constructs and Protein Expression
Kin1 was made byfusing Drosophila conventional kinesin truncated at posi-
tion 559 to a C-terminal eGFP and His6tag. Kin2 was engineered by swap-
ping the head and neck linker of mouse KIF3A into Kin1 (Figure 1C). See
Supplemental Experimental Procedures for details on cloning procedures
and sequences. All motors were expressed in bacteria and purified by Ni
column chromatography as described previously [15, 16].
Motility Assays
Bovine brain tubulin was purified and labeled with Cy5 (GE Healthcare) as
described previously [16–18]. Taxol-stabilized Cy5-labeled microtubules
were adsorbed onto the surface of flow cells, and the surfaces were
blocked with 2 mg/ml casein. Motility solution consisting of w20 pM
motors, 1 mM MgATP, 0.2 mg/ml casein, 10 mM Taxol, 20 mM D-glucose,
0.02 mg/ml glucose oxidase, 0.008 mg/ml catalase, and 0.5% v/v b-mer-
captoethanol in BRB80 (80 mM K-PIPES, 1 mM MgCl2, 1 mM EGTA [pH 6.8])
was then introduced. Single-molecule run lengths were visualized by TIRF
with a Nikon TE2000 microscope (603, 1.45 NA Plan Apo) equipped with
a 488 nm Ar ion laser for GFP excitation and a 633 nm He-Ne laser for
Cy5 excitation; experiments were performed at 26?C. Images were
captured with a Cascade 512 CCD camera (Roper Scientific), and acquisi-
tion and image analysis were carried out with MetaVue software (Molecular
Devices); pixel size was 71.0 nm. The duration and distance of single motor
runs were recorded manually. To ensure that all events were reliably
captured, we only analyzed events with a minimum run length of 250 nm,
and this minimum distance was subtracted from all runs (this assumes
that detachment probability is independent of the distance that the motor
has moved).
Supplemental Information
Supplemental Information includes Supplemental Results and Discussion,
three tables, four figures, and Supplemental Experimental Procedures and
can be found with this article online at doi:10.1016/j.cub.2010.03.065.
Acknowledgments
The authors thank V. Hariharan for work on molecular dynamics simulations
and N. Jones for protein purification assistance. This work was supported
by National Institutes of Health grant GM076476 to W.O.H.
Received: January 21, 2010
Revised: March 26, 2010
Accepted: March 29, 2010
Published online: May 13, 2010
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