Most MolecularMotor Proteins Are Members of the PLoop NTPase Superfamily

IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase
Superfamily
Eukaryotic cells contain three major families of motor proteins: myosins, kinesins, and dyneins. At first glance, these
protein families appear to be quite different from one another. Myosin, first characterized on the basis of its role in
muscle (Section 34.2.1), moves along filaments of the protein actin. Muscle myosin consists of two copies each of a
heavy chain with a molecular mass of 87 kd, an essential light chain, and a regulatory light chain. The human genome
appears to encode more than 40 distinct myosins; some function in muscle contraction and others participate in a variety
of other processes. Kinesins, which have roles in protein, vesicle, and organelle transport along microtubules, including
chromosome segregation, often consist of two copies each of a heavy chain and a light chain. The heavy chain is
approximately one-half the size of that for myosin. The human genome encodes more than 40 kinesins. Dynein powers
the motion of cilia and flagella in some eukaroytic cells, among other roles. Dyneins are enormous, with heavy chains of
molecular mass greater than 500 kd. The human genome appears to encode approximately 10 dyneins.
Comparison of the amino acid sequences of myosins, kinesins, and dyneins did not reveal significant relationships
between these protein families but, after their three-dimensional structures were determined, members of the myosin and
kinesin families were found to have remarkable similarities. In particular, both myosin and kinesin contain P-loop
NTPase cores homologous to those found in G proteins. Sequence analysis of the dynein heavy chain reveals it to be a
member of the AAA subfamily of P-loop NTPases that we encountered previously in the context of the 19S proteasome
(Section 23.2.2). Dynein has six sequences encoding such P-loop NTPase domains arrayed along its length. Thus, we
can draw on our knowledge of G proteins and other P-loop NTPases as we analyze the mechanisms of action of these
motor proteins.
34.1.1. A Motor Protein Consists of an ATPase Core and an Extended Structure
Let us first consider the structure of myosin. The results of electron microscopic studies of skeletal muscle myosin show
it to be a two-headed structure linked to a long stalk (Figure 34.2). As we saw in Chapter 33, limited proteolysis can be a
powerful tool in probing the activity of large proteins. Treatment of myosin with trypsin and papain results in the
formation of four fragments: two S1 fragments, an S2 fragment, also called heavy meromyosin (HMM), and a fragment
called light meromyosin (LMM; Figure 34.3). Each S1 fragment corresponds to one of the heads from the intact structure
and includes 850 amino-terminal amino acids from one of the two heavy chains as well as one copy of each of the light
chains. Examination of the structure of an S1 fragment at high resolution reveals the presence of a P-loop NTPasedomain core that is the site of ATP binding and hydrolysis (Figure 34.4).
Extending away from this structure is a long α helix from the heavy chain. This helix is the binding site for the two light
chains. The light chains are members of the EF-hand family, similar to calmodulin, although most of the EF hands in
light chains do not bind metal ions (Figure 34.5). Like calmodulin, these proteins wrap around an α helix, serving to
thicken and stiffen it. The remaining fragments of myosin
S2 and light meromyosin
are largely α helical, forming
two-stranded coiled coils created by the remaining lengths of the two heavy chains wrapping around each other (Figure
34.6). These structures, together extending approximately 1700 Å, link the myosin heads to other structures. In muscle
myosin, several LMM domains come together to form higher-order bundles.
Conventional kinesin, the first kinesin discovered, has a structure having several features in common with myosin
(Figure 34.7). The dimeric protein has two heads, linked by an extended structure. The size of the head domain is
approximately one-third of that of myosin. Determination of the three-dimensional structure of a kinesin fragment
revealed that this motor protein also is built around a P-loop NTPase core (Figure 34.8). The myosin domain is so much
larger than that of kinesin because of two large insertions in the myosin domain. For conventional kinesin, a region of
approximately 500 amino acids follows the head domain. Like the corresponding region in myosin, the extended part of
kinesin forms an α-helical coiled coil. Unlike myosin, the α-helical region directly adjacent to the head domain is not the
binding site for kinesin light chains. Instead, kinesin light chains, if present, bind near the carboxyl terminus.
Dynein has a rather different structure. As noted earlier, the dynein heavy chain includes six regions that are homologous
to the AAA subfamily of ATPase domains. Although no crystallographic data are yet available, the results of electron
microscopic studies and comparison with known structures of other AAA ATPases have formed the basis for the
construction of a model of the dynein head structure (Figure 34.9). The head domain is appended to a region of
approximately 1300 amino acids that forms an extended structure that links dynein units together to form oligomers and
interacts with other proteins.
34.1.2. ATP Binding and Hydrolysis Induce Changes in the Conformation and Binding
Affinity of Motor Proteins
A key feature of P-loop NTPases such as G proteins is that they undergo structural changes induced by NTP binding and
hydrolysis. Moreover, these structural changes alter their affinities for binding partners. Thus, it is not surprising that the
NTPase domains of motor proteins display analogous responses to nucleotide binding. The S1 fragment of myosin from
scallop muscle provides a striking example of the changes observed (Figure 34.10). The structure of the S1 fragment has
been determined for S1 bound to a complex formed of ADP and vanadate (VO4 3-), which is an analog of ATP, or, more
precisely, the ATP-hydrolysis transition state. In the presence of the ADP - VO4 3- complex, the long helix that binds the
light chains (hereafter referred to as the lever arm) protrudes outward from the head domain. In the presence of ADP
without VO4 3-, the lever arm has rotated by nearly 90 degrees relative to its position in the ADP - VO4 3- complex. How
does the identity of the species in the nucleotide-binding site cause this dramatic transition? Two regions around the
nucleotide-binding site, analogous to the switch regions of G proteins (Section 15.1.2), conform closely to the group in
the position of the γ-phosphate group of ATP and adopt a looser conformation when such a group is absent (Figure
34.11). This conformational change allows a long α helix (termed the relay helix) to adjust its position. The
carboxylterminal end of the relay helix interacts with structures at the base of the lever arm, and so a change in the
position of the relay helix leads to a reorientation of the lever arm.
The binding of ATP significantly decreases the affinity of the myosin head for actin filaments. No structures of myosin actin complexes have yet been determined at high resolution, so the mechanistic basis for this change remains to be
elucidated. However, the amino-terminal end of the relay helix interacts with the domains of myosin that bind to actin,
suggesting a clear pathway for the coupling of nucleotide binding to changes in actin affinity. The importance of the
changes in actin-binding affinity will be clear later when we examine the role of myosin in generating directed motion
(Section 34.2.4).
Analogous conformational changes take place in kinesin. The kinesins also have a relay helix that can adopt different
configurations when kinesin binds different nucleotides. Kinesin lacks an α-helical lever arm, however. Instead, a
relatively short segment termed the neck linker changes conformation in response to nucleotide binding (Figure 34.12).
The neck linker binds to the head domain of kinesin when ATP is bound but is released when the nucleotide-binding site
is vacant or occupied by ADP. Kinesin differs from myosin in that the binding of ATP to kinesin increases the affinity
between kinesin and its binding partner, microtubules. The properties of myosin, kinesin, and a heterotrimeric G protein
are compared in Table 34.1. Before turning to a discussion of how these properties are used to convert chemical energy
into motion, we must consider the properties of the tracks along which these motors move.
IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
Figure 34.2. Myosin Structure at Low Resolution. Electron micrographs of myosin molecules reveal a two-headed
structure with a long, thin tail. [Courtesy of Dr. Paula Flicker, Dr. Theo Walliman, and Dr. Peter Vibert.]
IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
Figure 34.3. Myosin Dissection. Treatment of muscle myosin with proteases forms stable fragments, including
subfragments S1 and S2 and light meromyosin. Each S1 fragment includes the head (shown in yellow and pink) from the
heavy chain and one copy of each light chain (shown in blue and orange).
IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
Figure 34.4. Myosin Structure at High Resolution. The structure of the S1 fragment from muscle myosin reveals the
presence of a P-loop NTPase domain (shaded in purple). An α helix that extends from this domain is the binding
site for the two light chains.
IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
Figure 34.5. Myosin Light Chains. The structures of the essential and regulatory light chains from muscle myosin are
compared with the structure of calmodulin. Each of these homologous proteins binds an α helix (not shown) by
wrapping around it.
IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
Figure 34.6. Myosin Two-Stranded Coiled Coil. The two α helices form left-handed supercoiled structures that spiral
around each other. Such structures are stabilized by hydrophobic residues at the contact points between the two
helices.
IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
Figure 34.7. Kinesin at Low Resolution. An electron micrograph of conventional kinesin reveals an elongated structure
with two heads at one end. The position of the light chains was confirmed through the use of antibody labels. [After N.
Hirokawa, K. K. Pfister, H. Yorifuji, M. C. Wagner, S. T. Brady, and G. S. Broom. Cell 56 (1989):867.]
IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
Figure 34.8. Structure of Head Domain of Kinesin at High Resolution. The head domain of kinesin has the structure
of a P-loop NTPase core (indicated by purple shading).
IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
Figure 34.9. Dynein Head-Domain Model. ATP is bound in the first of six P-loop NTPase domains (numbered) in this
model for the head domain of dynein. The model is based on electron micrographs and the structures of other
members of the AAA ATPase family.
IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
Figure 34.10. Lever-Arm Motion. Two forms of the S1 fragment of scallop muscle myosin. Dramatic conformational
changes are observed when the identity of the bound nucleotide changes from ADP-VO43- to ADP or vice versa,
including a nearly 90-degree reorientation of the lever arm.
IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
Figure 34.11. Relay Helix. A superposition of key elements in two forms of scallop myosin reveals the structural
changes that are transmitted by the relay helix from the switch I and switch II loops to the base of the lever arm. The
switch I and switch II loops interact with VO43- in the position that would be occupied by the γ-phosphate group of ATP.
The structure of the ADP - VO43- myosin complex is shown in lighter colors.
IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
Figure 34.12. Neck Linker. A comparison of the structures of a kinesin bound to ADP and bound to an ATP analog.
The neck linker (orange), which connects the head domain to the remainder of the kinesin molecule, is bound to
the head domain in the presence of the ATP analog but is free in the presence of ADP only.
IV. Responding to Environmental Changes
34. Molecular Motors
34.1. Most Molecular-Motor Proteins Are Members of the P-Loop NTPase Superfamily
Table 34.1. Effect of nucleotide binding on protein affinity
Bound to
Protein
Myosin (ATP or ADP)
NTP NDP