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Proteolytic mapping of kinesin/ncd-microtubule interface: nucleotide-dependent conformational changes in the loops L8 and L12

1998; Springer Nature; Volume: 17; Issue: 4 Linguagem: Inglês

10.1093/emboj/17.4.945

1460-2075

María C. Alonso, Jose van Damme, Joël Vandekerckhove, Robert A. Cross,

Advanced Electron Microscopy Techniques and Applications

Article15 February 1998free access Proteolytic mapping of kinesin/ncd-microtubule interface: nucleotide-dependent conformational changes in the loops L8 and L12 Maria C. Alonso Maria C. Alonso Molecular Motors Group, Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL UK Search for more papers by this author Jose van Damme Jose van Damme Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganck Straat 35, B-9000 Gent, Belgium Search for more papers by this author Joel Vandekerckhove Joel Vandekerckhove Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganck Straat 35, B-9000 Gent, Belgium Search for more papers by this author Robert A. Cross Corresponding Author Robert A. Cross Molecular Motors Group, Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL UK Search for more papers by this author Maria C. Alonso Maria C. Alonso Molecular Motors Group, Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL UK Search for more papers by this author Jose van Damme Jose van Damme Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganck Straat 35, B-9000 Gent, Belgium Search for more papers by this author Joel Vandekerckhove Joel Vandekerckhove Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganck Straat 35, B-9000 Gent, Belgium Search for more papers by this author Robert A. Cross Corresponding Author Robert A. Cross Molecular Motors Group, Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL UK Search for more papers by this author Author Information Maria C. Alonso1, Jose van Damme2, Joel Vandekerckhove2 and Robert A. Cross 1 1Molecular Motors Group, Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL UK 2Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ledeganck Straat 35, B-9000 Gent, Belgium *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:945-951https://doi.org/10.1093/emboj/17.4.945 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We used a battery of proteases to probe the footprint of microtubules on kinesin and ncd, and to search for nucleotide-induced conformational changes in these two oppositely-directed yet homologous molecular motors. Proteolytic cleavage sites were identified by N-terminal microsequencing and electrospray mass spectrometry, and then mapped onto the recently-determined atomic structures of ncd and kinesin. In both kinesin and ncd, microtubule binding shields a set of cleavage sites within or immediately flanking the loops L12, L8 and L11 and, in ncd, the loop L2. Even in the absence of microtubules, exchange of ADP for AMPPNP in the motor active site drives conformational shifts involving these loops. In ncd, a chymotryptic cleavage at Y622 in L12 is protected in the strong binding AMPPNP conformation, but cleaved in the weak binding ADP conformation. In kinesin, a thermolysin cleavage at L154 in L8 is protected in AMPPNP but cleaved in ADP. We speculate that ATP turnover in the active site governs microtubule binding by cyclically retracting or displaying the loops L8 and L12. Curiously, the retracted state of the loops corresponds to microtubule strong binding. Conceivably, nucleotide-dependent display of loops works as a reversible block on strong binding. Introduction The recently solved crystal stuctures of kinesin heavy chain (Kull et al., 1996) and its oppositely-directed homologue ncd (Sablin et al., 1996) revealed extensive structural similarities between the two molecules and also clear structural homology to the catalytic core of myosin and the RAS family of GTPases (Vale, 1996). The structural similarity suggests that the active sites of kinesin and ncd may undergo conformational changes, homologous to those that occur in RAS and myosin, particularly in response to the presence or absence of the gamma phosphate of ATP (Vale, 1996). How these putative motions in the motor active site are amplified globally in order to step the motor along the microtubule is, however, unknown. The mechanism of this so-called mechanochemical coupling is the major problem in the field. We describe here the use of a proteolytic version of the well-known DNA footprinting technique, to probe for nucleotide-induced conformational changes in kinesin and ncd, and to map the microtubule contact surfaces for the two motors in different nucleotides. The results support the conclusions of a recent alanine scanning study of the microtubule-binding face of kinesin (Woehlke et al., 1997). Our data extend the earlier work by providing information about the solvent-exposed face of kinesin, about both faces of ncd, and about nucleotide-dependent conformational effects at the microtubule recognition interfaces of both motors. Results The data are summarized in Table I. The dataset comprises 18 cleavage sites in kinesin and 19 in ncd. Digestions were made over a three-decades concentration range of each protease, in the presence and absence of superstoichiometric concentrations of microtubules, and in the presence of Mg-ADP or Mg-AMPPNP. Motor-ADP is a weakly microtubule-binding species (Kd ∼10–20 μM) and motor-AMPPNP is a strongly microtubule-binding species (Kd 98% in the supernatant in the absence of microtubules under these conditions, but pelletted ∼70% with the microtubules in the presence of AMPPNP, and ∼15% with microtubules in the presence of ADP. These binding controls are shown in Figure 2. Polypeptide separation, NH2-terminal sequencing and mass determination We sought to analyse only those digests that yielded relatively simple patterns of cleavage products, in which we could clearly discern which cleavage products were differentially protected by different nucleotides and/or microtubules. 10–20% SDS microslab gel electrophoresis and blotting on to PVDF membranes (Immobilon-P; Amersham) for N-terminal microsequencing was according to Matsudaira (1993). N-terminal sequences of cleavage products were determined initially by N-terminal microsequencing from Electroblots. C-terminal sequences were determined by HPLC fractionation of the digestion mixtures followed by on-line electrospray mass spectrometry. The identity of the HPLC peaks was reconfirmed by N-terminal sequencing, as follows. Protein fragments, generated by limited and controlled cleavage (as described above) were separated by reversed-phase HPLC on a C4 I.D., 2.1×50mm column (p/n 214TP5205, Vydac Separation Group Hesperia CA) using a 130A HPLC solvent delivery system (Applied Biosystems, Perkin Elmer). Solvent A was 0.1% trifluoroacetic acid (TFA) in water, while solvent B consisted of 0.09% TFA in water mixed with an isopropanol acetonitrile mixture (4:6) in a ratio 5:95 (by volume). A linear gradient from 5% B to 60% B in 90 min was generated at a flow of 80 μl/min at 50°C. Eluting polypeptides were detected by U.V. absorbancy at 214 nm. Peaks were collected manually in Eppendorf tubes and aliquots were taken for automated NH2-terminal sequence analysis using a pulsed-liquid sequenator model 477A equipped with a 120A phenylthiohydantoin amino acid analyser. The column eluate was separated with a flow splitter device directing 80% of the eluate into the UV detector and 20% into the ionisation chamber of a single quadrupole mass spectrometer (model Platform, Micromass, Manchester, UK). The instrument was equipped with an electrospray ion source and the m/z ratios were measured using a quadrupole analyser. The flow rate of the peptide carrier solvent at the inlet was ∼16 l/min. Droplet evaporation was achieved by heating the ion source at 60°C and with a stream of N2 gas at about 200 l/h. The mass spectrometer was scanned over m/z values ranging from 450 to 1550 at a rate of one scan every 7 s. Data acquisition was done in the centroid mode. 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