The Crystal Structure of the N-Terminal Region of BUB1 Provides Insight into the Mechanism of BUB1 Recruitment to Kinetochores

Summary The interaction of the central mitotic checkpoint component BUB1 with the mitotic kinetochore protein Blinkin is required for the kinetochore localization and function of BUB1 in the mitotic spindle assembly checkpoint, the regulatory mechanism of the cell cycle that ensures the even distribution of chromosomes during the transition from metaphase to anaphase. Here, we report the 1.74 Å resolution crystal structure of the N-terminal region of BUB1. The structure is organized as a tandem arrangement of three divergent units of the tetratricopeptide motif. Functional assays in vivo of native and site-specific mutants identify the residues of human BUB1 important for the interaction with Blinkin and define one region of potential therapeutic interest. The structure provides insight into the molecular basis of Blinkin-specific recognition by BUB1 and, on a broader perspective, of the mechanism that mediates kinetochore localization of BUB1 in checkpoint-activated cells.

12,661 eV Wild-type crystals diffracted to 1.74 Å, and selenomethionine-substituted crystals diffracted to 1.80 Å. Both wild-type and selenomethionine crystals belonged to space group C2 and contained two molecules of BUB1 in the asymmetric unit resulting in approximately 56% solvent content. All diffraction data were indexed, scaled and merged using HKL suite (Otwinowski and Minor, 1997). The initial sets of phases for BUB1 structure were obtained using a single anomalous diffraction technique using data from the selenomethionine-substituted crystals. The electron density maps prior to model building were subjected to a variety of density modification techniques including solvent flattening. Seleno sites were found and refined using PHENIX (Python-based Hierarchical ENvironment for Integrated Xtallography) (Adams et al, 2002). Refinement with REFMAC5 (Murshudov et al., 1997) produced high quality 2F o -F c and F o -F c electron density maps. The model was improved by rounds of refinement with REFMAC5 and manual rebuilding using COOT (Emsley and Cowtan, 2004) to an R factor of 18.9% and R free = 21.6% for all reflections in the test set with Bragg spacing between 50 and 1.74 Å. (Table I). The unbiased SAD-phased electron density maps were used to prevent model bias throughout refinement. Electron density maps obtained from these initial phases and native amplitudes clearly revealed all α-helices and most of the connecting loops and were readily traced. Ramachandran plot analysis using the programme RAMPAGE (Lovell et al., 2003) showed that all residues fall in the most favoured Ramachandran category, whilst PROCHECK (Laskowski et al., 1993) shows that 95.2% residues fall in the most favoured Ramachandran category and 4.8.0% in the allowed category.

Protein Analysis
Limited proteolysis, MALDI-TOF, amino acid composition analysis and N-terminal sequencing by the Edman degradation method were conducted at the PNAC facility (Department of Biochemistry, University of Cambridge).

Mass Spectrometry
Samples for nano-electrospray (nano-ES) mass spectrometry were prepared by dilution of the original 220 µM stock solution to a final concentration of 45 µM and buffer exchanged to 0.2 M ammonium acetate at pH 9 using Micro biospin chromatography columns. Mass spectra were recorded on a modified Q-TOF-2 (Sobott et al., 2002). Nano-ES capillaries were prepared in-house from borosilicate glass tubes of 1 mm outer diameter and 0.50 mm inner diameter (Harvard Apparatus, Kent) using a Flaming/Brown P-97 micropipette puller (Sutter Instrument Company, Intracel, Hertfordshire, UK), and gold coated using an S. E. sputter coater (Polaron, Energy Beam Sciences, MA). The capillary tips were cut under a stereo microscope to give inner diameters of 1-5 µm, and 2 µl of protein solution was loaded for sampling. The pressures and accelerating potentials in the mass spectrometer were optimized to remove adducts while preserving non-covalent interactions. The optimum experimental conditions were obtained with a cone voltage of 135 V and capillary voltage of 1.5 kV. External calibration of the spectra was achieved using solutions of caesium iodide, and the errors from calculated values for all reported masses are approximately 0.1%. Data acquisition and processing were performed using the MassLynx software (Micromass).

Small-Angle X-ray Scattering
Solution x-ray scattering data were collected at station 2.1, Synchrotron Radiation Source, Daresbury Laboratory UK, using a two-dimensional multiwire proportional counter at sample-to-detector distances of 1m and 4.25m and an x-ray wavelength of 1.54 Å with beam currents between 120 and 200 mA. Each sample was exposed for 25 min in 30 s frames. Frames at the beginning and the end of each data collection were compared to exclude the possibility of protein aggregation and/or radiation damage. The data reduction involved radial integration, normalization of the onedimensional data to the intensity of the transmitted beam, correction for detector artefacts, and subtraction of buffer scattering (OTOKO, SRS Daresbury). The q-range was calibrated with an oriented specimen of wet rat tail collagen (diffraction spacing 670 Å) and silver behenate (diffraction spacing 58.38 Å). Sc-BUB1  and  solutions at concentration ranging between 1 and 15 mg/ml (≈ 36-550 µM) were prepared in 0.05 M Tris-HCl, 0.15 M NaCl, pH 8.0 and analysed at 20 o C. The profiles collected at both camera lengths were merged so as to cover the momentum transfer interval 0.012 Å -1 < q < 0.778 Å -1 . The modulus of the momentum transfer is defined as q = 4 π sin Θ/λ, where 2Θ is the scattering angle and λ is the wavelength used. The maximum scattering angle corresponds to a nominal Bragg resolution of approximately 8 Å. The forward scattering intensity, radius of gyration R g , and the maximum particle dimension D max , and intraparticle distance distribution function [p(r)] were calculated from the scattering data using the indirect Fourier transform method program GNOM (Svergun, 1991). X-ray scattering predicted on the basis of the crystal structure of Sc-BUB1 (29-230) was compared to its conformation in solution using the program CRYSOL (Svergun et al., 1995) which simulates the scattering profile from atomic coordinates and provides a goodness-offit relating to the experimental data by inclusion of a hydration shell. For determinations of protein oligomerisation states, the following proteins were used as reference: BSA (67 kDa); Ovalbumin (43 kDa); Thaumatin I (22 kDa).

Circular Dichroism
Far-UV circular dichroism spectra were recorded on an AVIV 62-S spectropolarimeter (AVIV, New Jersey, USA) previously calibrated with camphorosulfonic acid and equipped with a temperature control unit. In all experiments, spectra were recorded at 20 o C in a 0.1 cm quartz cell using an average time of 1.0 s, a step size of 0.5 nm, 1-nm bandwidth, and averaged over 20 scans. The dependence of CD signal on protein concentration was calculated by triplicate using independent samples of concentrations ranging between 50 µg/ml to 600 µg/ml (i.e., ≈ 2-22 µM). After subtraction of the buffer baseline, the CD data were normalized and reported as molar residue ellipticity. For studies on protein stability as a function of pH, protein solutions were prepared in 0.05 M sodium acetate, pH 4 and 5; 0.05 M sodium phosphate buffer, pH 6, 7, 8 and 0.05 M sodium borate, pH 9 and 10. For thermal denaturation experiments, five unfolding curves were recorded upon heating from 5 to 95 o C at a rate of 1 o C/min, and 80 s accumulation time. The apparent melting temperature, Tm, was determined from differential melting curves of the function d[θ 222 ](T)/dt. The concentration of protein solutions was determined from amino acid composition analysis at the PNAC facility (Department of Biochemistry, University of Cambridge). Far-UV CD analysis of all proteins was carried out immediately after gel filtration chromatography.

Optimal Docking Area Analysis
The ODA method identifies protein surface patches with propensity for proteinprotein interactions on the basis of desolvation energy (Fernandez-Recio et al, 2005). The method calculates the effective desolvation energy for burying a continuous surface patch from the solvent according to atomic solvation parameters (Fernandez-Recio et al, 2004). The docking surface energy is calculated for patch residues that lie within a sphere of radius d (where d = 1, 2, . . ., 20 Å ) from the centre of coordinates of each residue side chain. The patch with lowest desolvation energy value is called an ODA. Significant ODAs are those with an optimal desolvation energy of less than -10.0 kcal/mol, and can be graphically displayed on the surface of the proteins by colouring each surface residue according to its ODA value (i.e., from red to blue indicating increasing propensities for a docking site). The ability of the ODA method to identify protein interaction sites has been validated on a set of three-dimensional structures of non-homologous proteins involved in non-obligate protein-protein interactions. Significant ODAs were found in half of the test proteins, and >80% of the ODAs identified corresponded to a known protein interaction site (Fernandez-Recio et al, 2005).

Yeast 2-Hybrid Analysis
The 2-hybrid analysis was performed according to the guidelines described by the manufacturer (MATCHMAKER 2-hybrid analysis kit, Clontech) using pGBT9 and pGAD424. The β-galactosidase filter assay was performed using the SFY526 strain that carried the GAL1-lacZ reporter according to conventional methods. (C) The Sc-BUB1 (29-230) mutant G146A-G148A shows a similar secondary structure as the native protein, suggesting its adopts a native-like fold. In all studies protein concentration was 600 µg/ml (i.e., ≈ 24 µM).

Figure S2. Analysis of Putative Protein-Protein Interaction Sites
Optimal Docking Area (ODA) of Sc-BUB1  . The absolute ODA scale, 0-40, is shown on the right upper corner; the colours, ranging from red to blue, represent increasing propensities for a binding site. The projection of ODA onto the protein surface shows that the region of highest propensity for protein-protein interactions involves α-helices H2, H4 and H6.