Ternary complex of Kif2A-bound tandem tubulin heterodimers represents a kinesin-13-mediated microtubule depolymerization reaction intermediate.

Trofimova D, Paydar M, Zara A, Talje L, Kwok BH, Allingham JS, Nat Commun 9(1):2628 (2018) PubMed

SASDCR9 – Kif2A-tubulin-DARP complex in the presence of AMP-PNP

Designed Ankyrin Repeat Protein D1
Kinesin-like protein KIF2A
Tubulin alpha-1B chain
Tubulin beta-2B chain
MWexperimental 259 kDa
MWexpected 266 kDa
VPorod 374 nm3
log I(s) 2.94×10-2 2.94×10-3 2.94×10-4 2.94×10-5
Designed Ankyrin Repeat Protein D1 Kinesin-like protein KIF2A Tubulin alpha-1B chain Tubulin beta-2B chain small angle scattering data  s, nm-1
ln I(s)
Designed Ankyrin Repeat Protein D1 Kinesin-like protein KIF2A Tubulin alpha-1B chain Tubulin beta-2B chain Guinier plot ln 2.95×10-2 Rg: 5.4 nm 0 (5.4 nm)-2 s2
(sRg)2I(s)/I(0)
Designed Ankyrin Repeat Protein D1 Kinesin-like protein KIF2A Tubulin alpha-1B chain Tubulin beta-2B chain Kratky plot 1.104 0 3 sRg
p(r)
Designed Ankyrin Repeat Protein D1 Kinesin-like protein KIF2A Tubulin alpha-1B chain Tubulin beta-2B chain pair distance distribution function Rg: 5.6 nm 0 Dmax: 19.5 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Designed Ankyrin Repeat Protein D1 Kinesin-like protein KIF2A Tubulin alpha-1B chain Tubulin beta-2B chain DAMMIF model

log I(s)
 s, nm-1
Designed Ankyrin Repeat Protein D1 Kinesin-like protein KIF2A Tubulin alpha-1B chain Tubulin beta-2B chain PDB model

Synchrotron SAXS data from solutions of Kif2A-tubulin-DARPin complex in presence of AMP-PNP in 20 mM HEPES, 1 mM MgCl2, 150 mM NaCl, pH 7.2 were collected at the BioSAXS G1 beam line at the Cornell High Energy Synchrotron Source (CHESS; Ithaca, NY, USA) using a CCD Finger Lakes CCD detector at a sample-detector distance of 1.5 m and at a wavelength of λ = 0.1267 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). 1200 successive 2 second frames were collected using size exclusion chromatography SAXS (SEC-SAXS; 3.70 mg/ml sample injection at 22°C).

For the SEC-SAXS analysis, a mixture of Kif2A, tubulin and DARPin was passed continuously through an X-ray sample cell via an in-line size exclusion column (GE Superdex 200 10/300 GL) at a flow rate of 1 ml/min. The column was pre-equilibrated with running buffer consisting of 20 mM HEPES, 150 mM NaCl, 1 mM MgCl2 pH 7.2. The protein sample was prepared at 25 μM tubulin, 27 μM DARP in and 20 μM Kif2A and injected into a 100 μL loop. Approximately 1200 two-second exposures were collected per sample, and 100 buffer profiles preceding the elution peaks were averaged and used for background subtraction.

Designed Ankyrin Repeat Protein D1 (DARPin D1)
Mol. type   Protein
Organism   synthetic construct
Olig. state   Monomer
Mon. MW   17.9 kDa
Sequence   FASTA
 
Kinesin-like protein KIF2A (Kif2A)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   47.8 kDa
 
UniProt   O00139 (153-553)
Sequence   FASTA
 
Tubulin alpha-1B chain (alpha tubulin)
Mol. type   Protein
Organism   Bos taurus
Olig. state   Dimer
Mon. MW   50.2 kDa
 
UniProt   P81947
Sequence   FASTA
 
Tubulin beta-2B chain (beta tubulin)
Mol. type   Protein
Organism   Bos taurus
Olig. state   Dimer
Mon. MW   49.9 kDa
 
UniProt   Q6B856
Sequence   FASTA
 
PDB code   6BBN