Helix capping interactions stabilize the N-terminus of the kinesin neck coiled-coil.

In an effort to understand how specific structural features within the kinesin neck, a region of the heavy chain located between the catalytic core and stalk domains, may contribute to motor processivity (an ability to remain attached to the microtubule filament), we have prepared several synthetic peptides corresponding to the neck region of human conventional kinesin and determined their secondary structure content and stability by CD spectroscopy. Our results show that the coiled-coil dimerization domain within the human kinesin neck region corresponds to residues 337 to 369 in solution, and thus is in excellent agreement with the recent X-ray crystallographic structures of rat brain kinesin. Further, we show that the first and last heptads of this region are absolutely critical for creating the high stability and association of the dimeric structure. Interestingly, addition of the 7 N-terminal neck-linker residues (330-336) to the coiled-coil domain significantly increased its stability (Delta GdnHCl midpoint of 1 M or an increase of approximately 1.5 kcal/mol), indicating that a strong structural link exists between the neck-linker and coiled-coil region. Subsequent high-resolution structural analysis of the residues located at the junction of the neck-linker and coiled-coil revealed the presence of the two helix capping motifs, the capping box (a reciprocal interaction of Thr 336 with Gln 339) and the hydrophobic staple (a hydrophobic packing interaction of Leu 335 with Trp 340). Substitution of Leu 335 and Thr 336 (the capping residues) with Gly completely eliminated the increased stability of the coiled-coil region observed in the presence of the neck-linker residues. Correspondingly, substitution of Trp 340, the first hydrophobic core d position residue of the coiled-coil, with an Ala residue resulted in a greater than expected decrease in stability and helicity of the coiled-coil structure. Subsequent analysis of the X-ray structure and substitution analysis of Lys 341 revealed that Trp 340 makes an important interchain hydrophobic interaction with Lys 341 of the opposite chain. Taken together these results reveal that a set of strong intra- and inter-chain interactions made up of the helix "capping box," "hydrophobic staple," and the newly identified "Leu-Trp-Lys sandwich" motifs stabilize the kinesin neck coiled-coil structure, thus preventing it from fraying and unfolding.

[1]  Jonathon Howard,et al.  Processivity of the Motor Protein Kinesin Requires Two Heads , 1998, The Journal of cell biology.

[2]  E. Mandelkow,et al.  The Crystal Structure of Dimeric Kinesin and Implications for Microtubule-Dependent Motility , 1997, Cell.

[3]  M. Steinmetz,et al.  A distinct 14 residue site triggers coiled‐coil formation in cortexillin I , 1998, The EMBO journal.

[4]  H. Morii,et al.  Identification of kinesin neck region as a stable alpha-helical coiled coil and its thermodynamic characterization. , 1997, Biochemistry.

[5]  Ronald D. Vale,et al.  Controlling Kinesin by Reversible Disulfide Cross-Linking , 2000, The Journal of cell biology.

[6]  J Engel,et al.  An autonomous folding unit mediates the assembly of two-stranded coiled coils. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Wei Jiang,et al.  Influence of the Kinesin Neck Domain on Dimerization and ATPase Kinetics* , 1997, The Journal of Biological Chemistry.

[8]  Ronald D. Vale,et al.  Engineering the Processive Run Length of the Kinesin Motor , 2000, The Journal of cell biology.

[9]  B. Meyer,et al.  Conformational preferences of a synthetic 30mer peptide from the interface between the neck and stalk regions of kinesin. , 2000, Biochemistry.

[10]  D. Hackney,et al.  Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Alan R. Fersht,et al.  Capping and α-helix stability , 1989, Nature.

[12]  R. Vale,et al.  The way things move: looking under the hood of molecular motor proteins. , 2000, Science.

[13]  L. Goldstein,et al.  The road less traveled: emerging principles of kinesin motor utilization. , 1999, Annual review of cell and developmental biology.

[14]  R. Hodges,et al.  Relationship of sidechain hydrophobicity and α‐helical propensity on the stability of the single‐stranded amphipathic α‐helix , 1995 .

[15]  J. Howard,et al.  The movement of kinesin along microtubules. , 1996, Annual review of physiology.

[16]  D. Hackney,et al.  Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli. , 1994, The Journal of biological chemistry.

[17]  Toshio Yanagida,et al.  Direct observation of single kinesin molecules moving along microtubules , 1996, Nature.

[18]  A. Hudspeth,et al.  Movement of microtubules by single kinesin molecules , 1989, Nature.

[19]  N. Kallenbach,et al.  The helix–coil transition in heterogeneous peptides with specific side‐chain interactions: Theory and comparison with CD spectral data , 1991, Biopolymers.

[20]  Luis Serrano,et al.  The hydrophobic-staple motif and a role for loop-residues in α-helix stability and protein folding , 1995, Nature Structural Biology.

[21]  D. Hackney,et al.  Highly processive microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains , 1995, Nature.

[22]  R. Hodges,et al.  Synthesis of a model protein of defined secondary and quaternary structure. Effect of chain length on the stabilization and formation of two-stranded alpha-helical coiled-coils. , 1984, The Journal of biological chemistry.

[23]  L. Goldstein,et al.  Bead movement by single kinesin molecules studied with optical tweezers , 1990, Nature.

[24]  K. Johnson,et al.  Pathway of ATP hydrolysis by monomeric and dimeric kinesin. , 1998, Biochemistry.

[25]  Ronald D. Vale,et al.  Role of the Kinesin Neck Region in Processive Microtubule-based Motility , 1998, The Journal of cell biology.

[26]  E. Mandelkow,et al.  The coiled-coil helix in the neck of kinesin. , 1998, Journal of structural biology.

[27]  C. Mant,et al.  Effect of the α-amino group on peptide retention behaviour in reversed-phase chromatography Determination of the pKa values of the α-amino group of 19 different N-terminal amino acid residues , 1993 .

[28]  Roger Cooke,et al.  A structural change in the kinesin motor protein that drives motility , 1999, Nature.

[29]  T. Yanagida,et al.  Movements of truncated kinesin fragments with a short or an artificial flexible neck. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[30]  E. Mandelkow,et al.  X-ray structure of motor and neck domains from rat brain kinesin. , 1997, Biochemistry.

[31]  George D. Rose,et al.  Sequence determinants of the capping box, a stabilizing motif at the N‐termini of α‐helices , 1994 .

[32]  E. Mandelkow,et al.  Image Reconstructions of Microtubules Decorated with Monomeric and Dimeric Kinesins: Comparison with X-Ray Structure and Implications for Motility , 1998, The Journal of cell biology.

[33]  M. Schliwa,et al.  Walking on two heads: the many talents of kinesin , 2000, Nature Reviews Molecular Cell Biology.

[34]  E. Taylor,et al.  Interacting Head Mechanism of Microtubule-Kinesin ATPase* , 1997, The Journal of Biological Chemistry.

[35]  Ronald D. Vale,et al.  Role of the kinesin neck linker and catalytic core in microtubule-based motility , 2000, Current Biology.

[36]  M. Schliwa,et al.  Reversal in the direction of movement of a molecular motor , 1997, Nature.

[37]  R. Hodges,et al.  Disulfide bond contribution to protein stability: positional effects of substitution in the hydrophobic core of the two-stranded alpha-helical coiled-coil. , 1993, Biochemistry.

[38]  C. Pace.  Determination and analysis of urea and guanidine hydrochloride denaturation curves. , 1986, Methods in enzymology.

[39]  W. Schief,et al.  Conformational changes during kinesin motility. , 2001, Current opinion in cell biology.

[40]  R. Hodges,et al.  The two‐stranded α‐helical coiled‐coil is an ideal model for studying protein stability and subunit interactions , 1992, Biopolymers.

[41]  R. Cross.  On the hand over hand footsteps of kinesin heads , 1995, Journal of Muscle Research & Cell Motility.

[42]  R. Hodges,et al.  Demonstration of Coiled-Coil Interactions within the Kinesin Neck Region Using Synthetic Peptides , 1997, The Journal of Biological Chemistry.

[43]  J. Correia,et al.  Sedimentation studies on the kinesin motor domain constructs K401, K366, and K341. , 1995, Biochemistry.

[44]  D. Shortle,et al.  Probing the determinants of protein folding and stability with amino acid substitutions. , 1989, The Journal of biological chemistry.

[45]  E. Mandelkow,et al.  The structural and mechanochemical cycle of kinesin. , 1998, Trends in biochemical sciences.

[46]  S. Endow,et al.  Determinants of kinesin motor polarity. , 1998, Science.

[47]  K. Johnson,et al.  Alternating site mechanism of the kinesin ATPase. , 1998, Biochemistry.

[48]  Susan P. Gilbert,et al.  Pathway of processive ATP hydrolysis by kinesin , 1995, Nature.

[49]  Ronald D Vale,et al.  The Directional Preference of Kinesin Motors Is Specified by an Element outside of the Motor Catalytic Domain , 1997, Cell.

[50]  R. Cortese,et al.  Circular dichroism study on the conformational stability of the dimerization domain of transcription factor LFB1. , 1991, Biochemistry.

[51]  J. Richardson,et al.  Amino acid preferences for specific locations at the ends of alpha helices. , 1988, Science.