4 research outputs found
Binding surfaces positioning in the active and inactive structure, and predicted molecular movement to yield inactive RB1. A., B.
<p>Relative orientation of the functional surfaces in the model of active, nonphosphorylated (A) and inactive, phosphorylated (B) RB1. Cartoon representation of Rb-NP with overlaid transparent surface with RB-N in light blue, RB-P in light-pink. The residues involved in docking LXCXE are shown in yellow, those forming the FXXXV motif are shown in purple and those for EXXXDLFD in cyan. The residues 346–355 which form a helix in unmodified RB-N but are disordered in inactive RB-NP are represented in dark grey <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058463#pone.0058463-Hassler1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058463#pone.0058463-Burke1" target="_blank">[21]</a>, amino acid groups involved in RB-N:P interphase interaction in the inactive conformation in red ([RB-N K136, D139, T140, T142, D145], [RB-P Q736, E737, K740, K729]) and orange [(RB-N L161, K164, L206-E209, L211-I213, F216, E282, E287, N290, N295] [RB-P Q736, E737, K740, K729]). <b>C., D.</b> Cartoon representation of active, nonphosphorylated, (C) and inactive, phosphorylated (D) RB1. RB-N B-fold is coloured in green and RB-P B-fold in purple (this different colour scheme has not been used elsewhere in the paper and is only used here for clarity). The residues 346–355 which are structured in unmodified RB-N and unstructured in inactive RB-NP are represented in dark grey. <b>E.</b> Predicted molecular movement yielding conformational RB1 inactivation. Note surfaces involved in binding LXCXE motif proteins in RB-P (salmon/pink) and the homologous surface involved in FXXXV binding in RB-N (cyan/blue) are collinear in the active (left) but not inactive form (right).</p
Characterisation of RB1 derivatives by small-angle X-ray scattering.
<p><b>A</b>. Experimental and calculated scattering patterns of ddRB<b>-</b>NP (1), MBP-ddRB<b>-</b>NP (2), ddRB<b>-</b>NP-MBP (3). Experimental SAXS data as black dots with black error bars. Lines (red) represent the fits from <i>ab initio</i> models shown in <b>C</b> (ddRB-NP), <b>D</b> (MBP-ddRB-NP) and <b>E</b> (ddRB-NP-MBP). The logarithm of the scattering intensity is plotted as a function of momentum transfer, s = 4πsin(θ/2)/λ where θ is the scattering angle and λ is the wavelength of the X-rays (1.5 Å). <b>B.</b> Distance distribution functions for ddRB-NP, MBP-ddRB-NP and ddRB-NP-MBP. <b>C.</b> Averaged <i>ab initio</i> models for ddRB-NP obtained using DAMMIN (grey semi-transparent spheres) and MONSA (RB-N blue spheres, RB-P red spheres) superimposed. The models are shown in two different views rotated by 90°. <b>D., E. </b><i>Ab initio</i> models of MBP-ddRB-NP (<b>D</b>) and ddRB-NP-MBP (<b>E</b>) obtained by MONSA. MBP is shown as green, ddRB-NP as grey spheres. The models are viewed as in <b>C</b>. <b>F.</b> Radius of gyration (<i>R<sub>g</sub></i>) distribution obtained by EOM for ddRB-NP. Distributions correspond to a random pool of 10.000 generated structures (blue) and the EOM optimized ensemble (red).</p
RB1 architecture and study design.
<p><b>A.</b> Schematic of RB1 domain structure. RB1 NH2-terminal domain (RB-N, light blue), RB1 pocket-domain (RB-P, raspberry), the position of the twin cyclin folds which form the core of each domain is indicated, RB1 C-terminal region (RB-C, yellow)<b>. B.</b> RB1 constructs used in this study indicating the range of amino-acids covered. In the MBP-RB-NP and MBP-ddRB-NP constructs maltose binding protein (MBP, green) is coupled to the N-terminus of the RB construct, while in ddRB-NP-MBP it is coupled to the C-terminus. In the ddRB-NP, MBP-ddRB-NP and ddRB-NP-MBP constructs two interstitial regions were deleted, corresponding to residues 250–269, the arginine-rich linker (R-linker) of the RB-N domain, and residues 579–643, corresponding to the pocket linker connecting RB-P domain pocket lobes (P-linker). The positions of cyclin-dependent kinase consensus sites in RB-NP are indicated, with sites retained in the ddRB-NP, MBP-ddRB-NP and ddRB-NP-MBP constructs bold and starred. <b>C.</b> Atomic models of the RB-N and RB-P domains, shown in ribbon representations. RB-N left, RB-P right. Cyclin-fold helixes are coloured, RB-N A-fold in cyan, RB-N B-fold in light blue, RB-P A-fold in dark salmon, RB-P B-fold in pink, other helixes and visible loops are shown as grey.</p
Single particle analysis of electron microscope images of MBP-ddRB-NP. A.-F.
<p>3D reconstruction of unmodified MBP-ddRB-NP. <b>A.</b>, <b>B</b>. Single particle reconstruction for unmodified MBP-ddRB-NP. Calculated density map of MBP-ddRB-NP, shown as surface representations in grey related by a 90<sup>o</sup> rotation. <b>C.</b> 3D reconstruction in mesh representation oriented as in <b>B</b> with the docked structures of the RB-N and RB-P domains (PDB codes 2QDJ and 3POM) shown as cartoons colour-coded as follows: RB-N domain lobe A -cyan, lobe B -light blue; RB-P domain lobe A -dark salmon and lobe B – pink. <b>D., E.</b> Segmented densities shown as solid surface representation with overlaid surface representation of the unmodified RB-NP 3D reconstruction in mesh. The density attributed to the MBP tag is shown in light green, that attributed to RB-N in light blue and to RB-P in light pink. <b>F.</b> Docked structures of the RB-N and RB-P domains (PDB codes 2QDJ and 3POM) without density mesh, shown as cartoons and colour-coded as in C. <b>G.–L.</b> 3D reconstruction of phosphorylated MBP-ddRB-NP. <b>G., H</b>. 3D reconstruction shown as a grey surface in two orthogonal views. <b>I.</b> 3D reconstruction in mesh representation oriented as in <b>H</b> with the docked structures of inactive RB-NP (PDB code 4ELJ) shown as cartoons colour-coded as follows:-. RB-N domain lobe A -cyan, lobe B -light blue; RB-P domain lobe A -dark salmon and lobe B – pink. <b>J., K.</b> Segmented densities shown as solid surface representation with overlaid surface representation of the 3D reconstruction in mesh<b>.</b> Same colour coding as in <b>D</b> and <b>E. L.</b> Docked structures of inactive RB-NP (PDB code 4ELJ) without density mesh, shown as cartoons colour-coded as in I.</p