Structure of a fast kinesin: implications for ATPase mechanism and interactions with microtubules
2001; Springer Nature; Volume: 20; Issue: 22 Linguagem: Inglês
10.1093/emboj/20.22.6213
ISSN1460-2075
Autores Tópico(s)Cellular Mechanics and Interactions
ResumoArticle15 November 2001free access Structure of a fast kinesin: implications for ATPase mechanism and interactions with microtubules Y.-H. Song Corresponding Author Y.-H. Song Max-Planck Unit for Structural Molecular Biology, D-22607 Hamburg, Germany Search for more papers by this author A. Marx A. Marx Max-Planck Unit for Structural Molecular Biology, D-22607 Hamburg, Germany Search for more papers by this author J. Müller J. Müller Max-Planck Unit for Structural Molecular Biology, D-22607 Hamburg, Germany Search for more papers by this author G. Woehlke G. Woehlke Department of Cell Biology, Ludwig-Maximilians-University, D-80336 München, Germany Search for more papers by this author M. Schliwa M. Schliwa Department of Cell Biology, Ludwig-Maximilians-University, D-80336 München, Germany Search for more papers by this author A. Krebs A. Krebs EMBL, D-69117 Heidelberg, Germany Search for more papers by this author A. Hoenger A. Hoenger EMBL, D-69117 Heidelberg, Germany Search for more papers by this author E. Mandelkow Corresponding Author E. Mandelkow Max-Planck Unit for Structural Molecular Biology, D-22607 Hamburg, Germany Search for more papers by this author Y.-H. Song Corresponding Author Y.-H. Song Max-Planck Unit for Structural Molecular Biology, D-22607 Hamburg, Germany Search for more papers by this author A. Marx A. Marx Max-Planck Unit for Structural Molecular Biology, D-22607 Hamburg, Germany Search for more papers by this author J. Müller J. Müller Max-Planck Unit for Structural Molecular Biology, D-22607 Hamburg, Germany Search for more papers by this author G. Woehlke G. Woehlke Department of Cell Biology, Ludwig-Maximilians-University, D-80336 München, Germany Search for more papers by this author M. Schliwa M. Schliwa Department of Cell Biology, Ludwig-Maximilians-University, D-80336 München, Germany Search for more papers by this author A. Krebs A. Krebs EMBL, D-69117 Heidelberg, Germany Search for more papers by this author A. Hoenger A. Hoenger EMBL, D-69117 Heidelberg, Germany Search for more papers by this author E. Mandelkow Corresponding Author E. Mandelkow Max-Planck Unit for Structural Molecular Biology, D-22607 Hamburg, Germany Search for more papers by this author Author Information Y.-H. Song 1, A. Marx1, J. Müller1, G. Woehlke2, M. Schliwa2, A. Krebs3, A. Hoenger3 and E. Mandelkow 1 1Max-Planck Unit for Structural Molecular Biology, D-22607 Hamburg, Germany 2Department of Cell Biology, Ludwig-Maximilians-University, D-80336 München, Germany 3EMBL, D-69117 Heidelberg, Germany *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2001)20:6213-6225https://doi.org/10.1093/emboj/20.22.6213 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We determined the crystal structure of the motor domain of the fast fungal kinesin from Neurospora crassa (NcKin). The structure has several unique features. (i) Loop 11 in the switch 2 region is ordered and enables one to describe the complete nucleotide-binding pocket, including three inter-switch salt bridges between switch 1 and 2. (ii) Loop 9 in the switch 1 region bends outwards, making the nucleotide-binding pocket very wide. The displacement in switch 1 resembles that of the G-protein ras complexed with its guanosine nucleotide exchange factor. (iii) Loop 5 in the entrance to the nucleotide-binding pocket is remarkably long and interacts with the ribose of ATP. (iv) The linker and neck region is not well defined, indicating that it is mobile. (v) Image reconstructions of ice-embedded microtubules decorated with NcKin show that it interacts with several tubulin subunits, including a central β-tubulin monomer and the two flanking α-tubulin monomers within the microtubule protofilament. Comparison of NcKin with other kinesins, myosin and G-proteins suggests that the rate-limiting step of ADP release is accelerated in the fungal kinesin and accounts for the unusually high velocity and ATPase activity. Introduction Kinesin motor proteins accomplish various intracellular microtubule-based transport processes (reviewed in Goldstein and Philp, 1999; Woehlke and Schliwa, 2000). Conventional kinesin from animals consists of two heavy and two light chains, while kinesin from the fungus Neurospora crassa lacks the light chains. The heavy chain of conventional kinesin is organized into three domains: the N-terminal motor domain, the central stalk and the C-terminal light chain-binding domain. It is often associated with membranous organelles and responsible for their transport within the cell. Deletion of kinesin in N.crassa causes retarded hyphal growth and loss of the 'Spitzenkörper', an organelle linked to cell morphogenesis (Seiler et al., 1997). The head domain of kinesin can be subdivided into a core motor domain of ∼325 residues, responsible for the ATPase activity and microtubule (MT) binding, and a 'linker' region (residues ∼325–340) connecting to the 'neck' (residues ∼340–370), the beginning of the coiled-coil stalk. A number of kinesin structures have been solved so far, including forward and reverse motors: human kinesin (HsKin; Kull et al., 1996), Ncd monomer and dimer (Sablin et al., 1996, 1998), rat kinesin monomer and dimer (RnKin; Kozielski et al., 1997; Sack et al., 1997; Muller et al., 1999), Kar3 (Gulick et al., 1998), Kif1A (Kikkawa et al., 2001), Eg5 (Turner et al., 2001) and Kar3 mutants (Yun et al., 2001). These analyses have shown that the core nucleotide-binding domain is related to that of myosins and G-proteins (Rayment, 1996; Kull et al., 1998). Structures of G-proteins and myosins complexed with different nucleotides and nucleotide analogues (Coleman and Sprang, 1999; Gulick et al., 2000; Holmes and Geeves, 2000) have revealed that all these proteins might share a similar mechanism of nucleotide hydrolysis (Vale and Milligan, 2000). The γ-phosphate-sensing regions of the G- and motor proteins are formed by three elements: the P-loop, switch 1 (Sw1) and switch 2 (Sw2). During nucleotide hydrolysis, the proteins undergo a conformational change within the Sw1 and Sw2 regions. The γ-phosphate forms a hydrogen bond with the amide group of a conserved glycine (G60 in Ras, G457 in myosin II; Hilgenfeld, 1995; Scheidig et al., 1995; Furch et al., 1999; Gulick et al., 2000). The corresponding residues in kinesins are G238 in N.crassa kinesin (NcKin) or G235 in RnKin. In myosin, the chain at this glycine undergoes a peptide flip in the ADP state so that the amide is turned away from the γ-phosphate-sensing region (Smith and Rayment, 1996). This small conformational change triggers a cascade of structural alterations. They propagate from the P-loop to the Sw1 and Sw2 regions until they end in a distant region of the enzyme, which can then induce the dramatic movement of domains comparable with that seen in EF-Tu or in the swing of the lever arm in myosin (Hilgenfeld, 1995; Houdusse et al., 2000). A key question is how this small difference in the active site can be transmitted to a distant location of the motor and modulate the affinity to the protein partners, e.g. MTs in the case of kinesin, F-actin for myosin, and the cofactors guanosine nucleotide exchange factor (GEF) or GTPase-activating protein (GAP) for the G-proteins (Hilgenfeld, 1995; Scheffzek et al., 1998; Holmes and Geeves, 2000). The case of kinesin is even more complicated because it is a highly processive motor and can take many steps along an MT before it dissociates. This is only possible if the two motor domains can communicate with each other. They achieve this by alternating site catalysis, whereby a transition in one head triggers a transition in the other (Hackney, 1996; Ma and Taylor, 1997; Gilbert et al., 1998). In order to understand the mechanism, it would be helpful to know the structure of different types of kinesin in different states of the ATPase cycle. Here we describe the structure of a fungal kinesin that moves ∼5 times faster than kinesins from animal sources (Steinberg and Schliwa, 1996; Seiler et al., 1997; Crevel et al., 1999), we show how it interacts with MTs and we discuss the basis of this high performance in comparison with other kinesins, myosin and G-proteins. Results General features of the structure The structure of the motor domain of kinesin from N.crassa was solved by molecular replacement, using RnKin as a template [Protein Data Bank (PDB) code 2kin; Sack et al., 1997]. The model contains all 355 residues of the motor domain with one MgADP and 128 water molecules (Table I; Figure 1A and B). The fold of the protein is similar to that of other kinesins, as expected from the sequence homology (55% identity between NcKin and RnKin). The central eight-stranded β-sheet is surrounded by six α-helices, three on either side (red in Figure 1A and B). Figure 1B represents a rear view of the motor domain as seen from the inside of the MT, with the green elements (β5–L8, L11, α4–L12–α5) facing a β-tubulin subunit. The small lobe of NcKin (strands β1a, β1b and β1c) differs from that of RnKin by an insertion of four amino acids between β1b and β1c. There is also a large displacement in the MT-interacting region around β5a, β5b and the connecting loops (L8a and L8b) (Figure 1C and D). Helix α1 contains a bend which is common to all known kinesin structures. Helix α2 is interrupted through the 11 residue bulge loop L5, the longest interruption within α2 found in kinesin members. Interestingly, the greatest deviations between NcKin and RnKin are found in those regions that have been proposed to change conformation during the ATPase cycle, namely Sw1, Sw2 and the MT-binding surface. Helix α3 is 23 residues long, compared with 16 residues in RnKin, resulting in a 35 Å long helix. Helix α3a comprises only a half turn, much shorter than in other kinesin structures (Figure 1C and D). This alteration is remarkable as these residues belong to the Sw1 region and form part of the active site. L11, located close to both the nucleotide- and MT-binding regions, is ordered and thus visible, in contrast to other kinesin structures. Another region of interest is the transition from the core motor domain to the stalk (end of α6, residue ∼325 for RnKin and ∼329 for NcKin). This region was mostly disordered in the HsKin structure (Kull et al., 1996), but in RnKin showed an extended linker (β9 and β10, residues ∼325–339) and the beginning of the α-helical neck (from ∼339 onwards), which leads into the coiled-coil stalk domain and causes dimerization. In NcKin, the last 22 residues show weak density. The linker has an extended structure, similar to RnKin, but there is no evidence for an α-helical neck (Figure 1D, bottom right). This corresponds to a lower predicted helix-forming propensity in the initial segment of the neck region, compared with RnKin (Thormahlen et al., 1998). This region undergoes large structural transitions during the movement of kinesin and is probably intrinsically mobile (Rice et al., 1999). Figure 1.(A and B) Ribbon diagram of the NcKin355 showing the overall fold. (A) Front view; (B) rear view, from the MT-binding surface. It is a globular structure with a central β-sheet of eight strands (β1–β8) and three α-helices on either side. α-helices (α0–α3, α6) are in red, and β-strands are in blue. The elements putatively involved in MT binding are coloured in green (α4–L12–α5, L11). ADP is shown as a space-filling model. (C and D) Comparison between the structures of NcKin and kinesin from rat brain. (C) Front view; (D) rear view. The structures of the NcKin and RnKin monomer (PDB code: 2kin) were superimposed by aligning the residues of the P-loops (NcKin 81–92, RnKin 97–92). Only regions with substantial variations in structure are shown in colour, NcKin in red and RnKin in green. L11 is ordered in the NcKin structure and elongates the Sw2 helix (α4) to α4a and the strand β7 to β7a. Helix α3 becomes longer (three turns more than in the RnKin structure) and α3a shorter. The interruption of the helix α2 is also longer. The linker region β9–β10 and following helical neck α7 of RnKin adopt a more disordered conformation in NcKin. Download figure Download PowerPoint Table 1. Data collection and refinement statistics Space group P212121 Unit cell parameters a = 51.97 Å b = 72.73 Å c = 84.93 Å Resolution range 30−2.3 Å Reflections, observed/unique 138 093/14 829 Completeness, overall/last shell 99.8%/100% Rsyma, overall/last shell 9.9%/31.9% Refinement statistics Resolution range 30−2.3 Å Rcryst (%)b/Rfree (%)c 21.9%/25.6% Coordinate error, Luzzati/Sigmaa (Å) 0.32/0.21 R.m.s. deviations bond lengths (Å) 0.006 bond angles (°) 1.3 torsion angles (°) 23.3 improper torsion angles (°) 0.83 a Rsym = ΣhklΣi|Ii − |/ΣhklΣi where Ii is the ith measurement of the reflection intensity I and is the weighted mean of all measurements of I. b Rcryst = Σhkl|Fobs − Fcalc|/ΣFobs where Fobs and Fcalc are the observed and the calculated structure factors, and the summation is over 90% of reflections used for model refinement. c Rfree, as for Rcryst except summed only over the 10% of reflections not used for model refinement. The unique structure of loop 11 and the switch 2 region of NcKin A remarkable feature of NcKin is the ordered structure of L11 in the Sw2 region following strand β7. In part, it forms a secondary structure element resulting in the extension of β7 to β7a, which in turn follows a loop and continues along the novel helix α4a, extending the Sw2 helix α4 by 1.5 turns (Figure 2). This region is disordered in other kinesin structures, except possibly in the initial Ncd structure where an antiparallel sheet, β7a and β7b, was reported (Sablin et al., 1996). The sequence at the beginning of L11 (235-DLAGSEKVGKT-245 in NcKin) is highly conserved throughout all kinesins and contains the epitope of a widely used antibody (LAGSE motif, shown in bold; Sawin et al., 1992). The sequence also contains a GxxxxGKT motif (underlined above) which is typical of nucleotide-binding P-loops and prompted speculations about a second nucleotide-binding site (Steinberg and Schliwa, 1996). However, the structure of L11 shows that this region has no direct contact with the phosphate of the bound nucleotide, in contrast to the functional P-loop, residues 88–95; and other kinesins such as RnKin contain a serine instead of glycine at position 243, thus breaking the P-loop motif. Interestingly, there is a mutant of NcKin with a Gly243Ser exchange, as in the RnKin sequence (Figure 3). This mutant displays a 25% reduced growth rate of hyphae and a reduced MT gliding velocity (only 1.9 versus 2.6 μm/s; U.Henningsen and M.Schliwa, unpublished). It therefore appears that the nature of residue 243 is critical for kinesin velocity, and that structural transitions in L11 are important for the ATPase cycle. Figure 2.Comparisons between the Sw2 structures of NcKin (black) and Ncd (grey) in ribbon representations. The orientation of the view is the same as in Figure 1. The switch 2 helix α4 and the strand β are somewhat extended into the L11 region. Download figure Download PowerPoint Figure 3.Loop 11 and the growth rate of hyphae. (A) Conformation of L11 (residues 240–253), starting at strand β7a and ending at the short helix α4a, an extension of the switch 2 helix α4. Residue G243 is partly responsible for the high velocity of NcKin. (B and C) Growth of hyphae observed by phase-contrast microscopy. There is a time delay of 3 min between micrographs (B) and (C); the growth rate is 8.5 μm/min. Note the dark 'Spitzenkörper' at the tip. Download figure Download PowerPoint The nucleotide-binding site The nucleotide-binding pocket is formed by four elements, N1–N4 (Kull et al., 1996). They include highly conserved regions: (i) L1 (N4, motif 14-RxRP-17), which interacts with the base; (ii) the phosphate-binding loop (N1 or P-loop, motif 88-GxxxxGKT-95); (iii) Sw1 (N2) including the region from α3a to β6 (motif 202-NxxSSR-207); and (iv) Sw2 (N3) including the region after β7 (motif 235-DLAGSE-240) and L11 (Figure 4A). Y96 at the end of the P-loop mediates a stacking interaction with the purine ring of the adenine base on one side, which corresponds to the histidine residue in RnKin (Muller et al., 1999). On the opposite side, the base makes a hydrophobic interaction with the ring of P16 and the hydrophobic portion of the side chain of R15. The adenine base is coordinated further through a water molecule (S123), which mediates a hydrogen bond between the base nitrogen N6 to the carbonyl oxygens of C59 and M57. The ribose oxygen O2′ binds a water molecule, which mediates a hydrogen bond to T101, a residue of the bulge loop L5 between α2a and α2b. This is of special interest because such a coordination of the ribose moiety has not been reported in any other known kinesin structure. The coordinating T101 is an inserted residue in comparison with the sequence of RnKin and HsKin (Table II) and might play a role in the fast NcKin ATPase cycle. In the case of Ncd, a mutation of the conserved G446 to arginine in L5 causes complete loss of the function of the protein (Endow and Komma, 1997). Figure 4.(A) Nucleotide-binding site and the coordination of ADP. Stereo diagram of the active site with bound ADP with its electron density, calculated as an omit map (Fo − Fc), and contoured at 1.5σ (see text). (B–D) The size of the nucleotide-binding pocket (stereo views). (B) Ribbon representation of the structure of NcKin. (C) Surface potential representation of the nucleotide-binding pocket of NcKin. (D) Kar3. The surface potentials are contoured according to the surface charge, with red denoting acidic and blue basic regions. The active site of NcKin is the most spacious among the known structures of kinesin motors. Figures were prepared using the programs SPOCK (Christopher, 1998) and Raster3d (Merrit, 1994); see Supplementary data. Download figure Download PowerPoint Table 2. Alignments of β-hairpin loop L5 NcKin G100 TSIDDPDGR G110 RnKin G98 KLHDPQLM G107 HsKin G97 KLHDPEGM G106 Ncd G446 VPESV G452 Kar3 N486 PGD G490 The bulge loop L5 of the plus motors is generally longer than that of the minus motors. Since L5 is close to the P-loop and is also part of the nucleotide 'entrance', the length of this loop might influence the ATPase cycle. The bridge oxygen between the α- and β-phosphate builds a hydrogen bond to the amide nitrogen of G93 at the end of the P-loop. The Mg2+ ion is coordinated octahedrally by six oxygen atoms also found in the nucleotide-binding pocket of RnKin (Muller et al., 1999). This includes a substrate oxygen atom on the β-phosphate, the side chain oxygen of S95 and four water molecules. Access to nucleotide-binding pocket and switch 1 region The terms 'open' and 'closed' applied to the pockets of the active site originate from the G-protein and myosin structures and refer to the movement of Sw1 and Sw2 relative to each other. The 'closed' conformation is usually found in the presence of ATP or GTP and their analogues (Mittal et al., 1996; Coleman and Sprang, 1999; Gulick et al., 2000). The distance between the conserved glycine at the beginning of the P-loop (G86 in RnKin) and the conserved glycine in the LAGSE motif of Sw2 (G235 in RnKin) has been taken as a measure for the openness of the nucleotide-binding pocket (Sack et al., 1999). In NcKin, this distance (G88–G238; 5.99 Å) is almost the same as in the structure of RnKin (5.87 Å) and Kar3 (5.82 Å). This means that the nucleotide-binding pocket of NcKin and the other kinesins can be considered as 'closed' even when the bound nucleotide is ADP, not ATP. Considering that the ATPase cycle contains several subreactions, the single criterion of the G88–G238 distance is likely to oversimplify the movements of Sw1 and Sw2. We therefore looked for other criteria to describe the accessibility of the nucleotide pocket in kinesin. The entrance of the nucleotide is at loops L1–L3–L5 and L9 (Figure 4B, green). The size of the pocket is determined critically by the position of L9, which belongs to the Sw1 region. In the case of Kar3, this loop is bent so far inwards that the active site becomes much smaller (Gulick et al., 1998). Therefore, the distance between the conserved glycine of the P-loop (G88) and the conserved asparagine of L9 in Sw1 (N202) would be an alternative measure of the openness of the pocket. In NcKin, the two residues (G88 and N202) are 22.7 Å apart, while for RnKin the corresponding distance (between G86 and N199) is 15.8 Å. Thus, this distance distinguishes between kinesin family members with different activities. Note that the minus motors (Ncd and Kar3) form a bulge loop between α3 and α3a (Figure 5B), whereas the plus motors do not (Figure 5A). Thus, besides the short distance between the residues glycine and asparagine compared with NcKin, the bulge loop in the reverse motors tightens the entrance of the active site even further. Figure 5.Comparisons of the Sw1 structure of different proteins of the kinesin family. (A) Superposition of the Sw1 structures of NcKin (red) versus RnKin (green) and HsKin (cyan). (B) Superposition of NcKin (red) versus Ncd (light blue) and Kar3 (blue). The orientation of this model is rotated 90° around the y-axis and 30° around the x-axis compared with the view in Figure 1A and B. Displacement of Sw1 as an essential step for nucleotide exchange. (C) Superposition of Kar3 and NcKin (only the P-loop and Sw1 are shown). The displacement of Sw1 (A197 in NcKin and A587 in Kar3) between the two structures is ∼15 Å. (D) Superposition of Ras-GTP and of the Ras–Sos structure. The displacement of Sw1 is also ∼15 Å, measured by the distance at residue Y32 (Boriack-Sjodin et al., 1998). Download figure Download PowerPoint Salt bridges involved in nucleotide binding and switching The structures of myosins in different nucleotide states, including ADP, ATP, AMP-PNP and ADP-VO4, revealed that the movement of Sw1 and Sw2 was accompanied by the reversible formation of critical salt bridges. Thus, the salt bridge R238–E459 is formed in myosin only in a closed-cleft structure complexed with ADP-VO4 (Smith and Rayment, 1996). We refer to the corresponding salt bridge as the inter-switch salt bridge type-A (SbA). Another important interaction is the hydrogen bond between the γ-phosphate and the amide of G457 observed in myosin II. To gain some insight into the ATPase mechanism of kinesin, we compared salt bridges and hydrogen bonds around the active site of NcKin with those of known kinesin structures (Figure 6). In all structures, at least one salt bridge was found, mostly between Sw1 and Sw2 (Table III). A unique feature of NcKin is that there are three inter-switch salt bridges (Figure 6). (i) The salt bridge E240–R207 is of the same type as that in RnKin and myosin complexed with ADP-VO4 (SbA). (ii) The salt bridge between K257 (Sw2) and E204 (Sw1) in NcKin is of a new type, termed SbC. It can only be observed in NcKin because L11 is ordered in NcKin. This interaction is probably important for the stabilization of L11. (iii) The third salt bridge is between D235 and R194, named SbB. These residues are preserved throughout the family of kinesin proteins. The residues of SbB are connected with the Mg2+ ion via a water-mediated hydrogen bond. This means that this salt bridge is likely to play a role in the dissociation of the Mg2+ ion from the active site. It was shown that the disruption of the Mg2+ coordination is the key step for dissociation of the nucleotide GDP in P21ras GTPase, where the GEF-mediated displacement of the Sw1 region of ∼15 Å is involved (Figure 5D) (Mittal et al., 1996; Boriack-Sjodin et al., 1998). Such a movement of Sw1 can be observed in NcKin in comparison with Sw1 of the other kinesins (Figure 5A and B). In the case of HsKin (Kull et al., 1996), only one intra-Sw1 salt bridge (E199–R203) can be found, here termed 'type-D' (SbD). The residues for the three types of salt bridge are preserved in all kinesins; however, the sets of salt bridges actually found in each structure are different (Figure 6B). Figure 6.Comparisons of salt bridges at the γ-phosphate-sensing region. (A) Stereo view of the superposition of NcKin (dark colour) and RnKin (pale colour). In NcKin, there are three inter-switch salt bridges and in RnKin there is only one. The salt bridge E97–K188 of RnKin cannot be formed in NcKin because at the corresponding positions there are no charged residues, which are Met99 and Gly191, respectively. (B) Summary of all known salt bridges of known kinesin structures. Download figure Download PowerPoint Table 3. Salt bridges NcKin RnKin Ncd Myosin (Dictyo) SbA E240(Sw2)–R207(Sw1) E237(Sw2)–R204(Sw1) D580(Sw2)–R539(Sw1) E459(Sw2)–R238(Sw1) SbB D235(Sw2)–R194(Sw1) D454(Sw2)–K241(Sw1) SbC K257(Sw2)–E204(Sw1) SbD E97(P-loop)–K188(Sw1) D445(P-loop)–R539 Interaction between the motor domain of NcKin and microtubules We investigated the NcKin355-decorated MTs by cryo-electron microscopy of unstained vitrified samples and calculated the three-dimensional image reconstructions of the complexes. Since high resolution structures are available both for tubulin (Nogales et al., 1999) and NcKin (this work), one can fit the molecules into the electron density derived from cryo-electron microscopy (Figure 7A). The MT is shown with its plus end (= fast growing end) towards the top of the page so that the orientation of the attached kinesin is upside down, compared with Figure 1 (for details on the identification see Song and Mandelkow, 1993; Hoenger et al., 1998). The bulk of the interaction involves the C-terminal part of β-tubulin (helices H11 and H12) and the region α4–L12–α5 of NcKin. The interaction appears to be mediated mainly by electrostatic force. There are several negatively charged residues in helix H12 of β-tubulin (E420, D427 and E431 numbered according to the bovine β-tubulin sequence) which can accomplish electrostatic interactions with the positively charged residues in L12 and α5 of kinesin (K276 and R283). Indeed, the equivalent residue of R283 has been reported to have a pronounced influence on the binding and activity of HsKin (Woehlke et al., 1997), and positively charged insertions into L12 of kinesin ('K-loop') increase the interactions with MTs so that even monomeric kinesins (Kif1A) can become processive (Kikkawa et al., 2000). The β5–L8 region (red, Figure 7A and B) interacts with β-tubulin, as noted earlier (Sack et al., 1999). Of special interest is the position of L11, which is visible in the NcKin structure. Its main interaction appears not to be primarily with the central β-tubulin subunit (Figure 7B, green) but with the next α-tubulin subunit below (blue, towards the MT minus end) at the loop between helices H11 and H12. In addition, it is possible that L11 of kinesin interacts with the C-terminal region of β-tubulin in the adjacent protofilament (see Supplementary data available at The EMBO Journal Online). Since L11 of kinesin is in the vicinity of the γ-phosphate-sensing region, its interaction with α-tubulin probably plays a role during ATP hydrolysis. This might be one of the key elements in the MT-activated kinesin ATPase, consistent with the effect of mutant G243S (Figure 3). A third region of interest is the linker/neck region of kinesin. Although it is structurally not well defined, it emerges near the upper end of β-tubulin, on the right side of the protofilament (upper right corner in Figure 7A and B), and could thus interact with the C-terminal region of the next α-tubulin in the plus direction. This would explain why the opposite charges in kinesin's neck and tubulin's C-terminus are important for processive movement (Thorn et al., 2000; Wang and Sheetz, 2000). Figure 7.Image reconstructions of MTs decorated with NcKin and interactions between them. Stereo views. (A) View from the side of a protofilament. Protein structures docked into the electron density are shown in green (β-tubulin), blue (α-tubulin) and grey/red (kinesin). The bound ADP is visible on the lower tip of kinesin. The bulk of the electron density of kinesin lies over a β-tubulin subunit, but there is some overlap with other subunits, especially the two flanking α-tubulin subunits within the same protofilament. (B) View from the side of a tubulin protofilament (i.e. tangential to the MT surface). Selected elements in the interacting regions are highlighted, α-helices as tubes, β-strands as ribbons. In NcKin, the elements L11, helix α4 (Sw2 region) and the region up to the C-terminus are purple, β4 and the β5/loop8 region (Sw1) are red. The tubulin subunits are blue (α-tubulin) and green (β-tubulin). The tubes show the near C-terminal helices H11 and H12, as well as helices H4, H5, H8 and adjacent loops. The C-terminal tail of tubulin (not visible in the structure due to disorder) has been drawn in arbitrary conformations in order to highlight potential interactions with kinesin (see text). Download figure Download PowerPoint Discussion Relationship of the Nckin structure to other kinesin motor domains The main function of NcKin is in cell morphogenesis, especially in the longitudinal growth of the hyphae and the formation of a 'Spitzenkörper' (Seiler et al., 1997). NcKin is the fastest kinesin known so far (velocity ∼2.6 μm/s in vitro; Kirchner et al.,
Referência(s)