Control of the structural stability of the tubulin dimer by one high affinity bound magnesium ion at nucleotide N-site.


Abstract

Tubulin liganded with GTP at the N-site in the alpha-subunit and with GDP at the E-site in the beta-subunit (GDP-tubulin) reversibly binds one high affinity Mg2+ cation (Kb = 1.1 x 10(7) M-1), whereas tubulin liganded with GTP at both subunits (GTP-tubulin) binds one more high affinity Mg2+. The two cation binding loci are identified as nucleotide sites N and E, respectively. Mg2+ at the N-site controls the stability and structure of the alphabeta-tubulin dimer. Mg2+ dissociation is followed by the slow release of bound nucleotide and functional inactivation. Mg2+ bound to the N-site significantly increases the thermal stability of the GDP-tubulin dimer (by 10 degrees C and approximately 50 kcal mol-1 of experimental enthalpy change). However, the thermal stability of Mg2+-liganded GDP- and GTP-tubulin is the same. Mg2+ binding to the N-site is linked to the alphabeta-dimer formation. The binding of Mg2+ to the alpha-subunit communicates a marked enhancement of fluorescence to a colchicine analogue bound to the beta-subunit. Colchicine, in turn, thermally stabilizes Mg2+-depleted tubulin. The tubulin properties described would be simply explained if the N-site and the colchicine site are at the alpha-beta dimerization interface. It follows that the E-site would be at the beta-end of the tubulin dimer, consistent with the known functional role of the E nucleotide gamma-phosphate and coordinated cation controlling microtubule stability. Study holds ProTherm entries: 10909, 10910, 10911, 10912, 10913, 10914, 10915, 10916, 10917, 10918, 10919, 10920, 10921, 10922, 10923, 10924, 10925, 10926, 10927 Extra Details: cation binding loci; thermal stability; alpha beta-dimer formation;

Submission Details

ID: 92WAGzvb

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:41 p.m.

Version: 1

Publication Details
Menéndez M;Rivas G;Díaz JF;Andreu JM,J. Biol. Chem. (1998) Control of the structural stability of the tubulin dimer by one high affinity bound magnesium ion at nucleotide N-site. PMID:9417061
Additional Information

Study Summary

Number of data points 38
Proteins Tubulin--tyrosine ligase
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dHcal ionic:Mg++: 160 micro M ; Experimental Assay: Tm ionic:Mg++: 160 micro M ; Experimental Assay: dHcal ionic:Mg++: 2.3 micro M ; Experimental Assay: Tm ionic:Mg++: 2.3 micro M ; Experimental Assay: dHcal ionic:Mg++: 280 micro M ; Experimental Assay: Tm ionic:Mg++: 280 micro M ; Experimental Assay: dHcal ionic:Mg++: 4.5 micro M ; Experimental Assay: Tm ionic:Mg++: 4.5 micro M ; Experimental Assay: dHcal ionic:Mg++: 927 nM ; Experimental Assay: Tm ionic:Mg++: 927 nM ; Experimental Assay: dHcal ionic:Mg++: 500 nM ; Experimental Assay: Tm ionic:Mg++: 500 nM ; Experimental Assay: dHcal ionic:Mg++: 182 nM ; Experimental Assay: Tm ionic:Mg++: 182 nM ; Experimental Assay: dHcal ionic:Mg++: 30 nM ; Experimental Assay: Tm ionic:Mg++: 30 nM ; Experimental Assay: dHcal ionic:Mg++: 75 nM ; Experimental Assay: Tm ionic:Mg++: 75 nM ; Experimental Assay: dHcal ionic:Mg++: 40 nM ; Experimental Assay: Tm ionic:Mg++: 40 nM ; Experimental Assay: dHcal ionic:Mg++: 50 nM ; Experimental Assay: Tm ionic:Mg++: 50 nM ; Experimental Assay: dHcal ionic:Mg++: 35 nM ; Experimental Assay: Tm ionic:Mg++: 35 nM ; Experimental Assay: dHcal ionic:Mg++: 25 nM ; Experimental Assay: Tm ionic:Mg++: 25 nM
Libraries Mutations for sequence MYTFVVRDENSSVYAEVSRLLLATGHWKRLRRDNPRFNLMLGERNRLPFGRLGHEPGLMQLVNYYRGADKLCRKASLVKLIKTSPELAESCTWFPESYVIYPTNLKTPVAPAQDGIHPPLHSSRTDEREFFLASYNRKKEEGEGNVWIAKSSAGAKGEGILISSDATELLDFIDNQGQVHVIQKYLERPLLLEPGHRKFDIRSWVLVDHQFNIYLYREGVLRTASEPYHMDNFQDKTCHLTNHCIQKEYSKNYGKYEEGNEMFFEAFNRYLTSALNITLESSILLQIKHIIRSCLMSVEPAISTKHLPYQSFQLFGFDFMVDEELKVWLIEVNGAPACAQKLYAELCQGIVDIAIASVFPPPDAEQQPPQPATFIKL

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5J2U 2016-08-17 2.5 Tubulin-MMAF complex

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.0 Tubulin--tyrosine ligase Q9QXJ0 TTL_RAT
96.4 Tubulin--tyrosine ligase P38160 TTL_PIG
93.1 Tubulin--tyrosine ligase P38585 TTL_MOUSE
93.9 Tubulin--tyrosine ligase Q8NG68 TTL_HUMAN
100.0 Tubulin--tyrosine ligase P38584 TTL_BOVIN