Structure and functional analysis of the IGF-II/IGF2R interaction

Transcription

1 Structure and functional analysis of the IGF-II/IGF2R interaction James Brown 1, Carlie Delaine 2, Oliver J. Zaccheo 3, Christian Siebold 1, Robert J. Gilbert 1, Gijs van Boxel 1, Adam Denley 2, John C. Wallace 2, A. Bassim Hassan 3, Briony E. Forbes 2, E. Yvonne Jones 1 1 Cancer Research UK Receptor Structure Research Group, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7BN, UK 2 School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, 5005, Australia 3 Tumour Growth Control Group, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK Supplementary Information Glycosylation status of IGF2R fragments All IGF2R fragments were expressed in Lec Chinese hamster ovary cells which produce EndoH-sensitive Man 5 GlcNAc 2 glycosylation (Stanley, 1989). If desired, these sugars were removed by overnight incubation at 37 C in 150 mm MES ph 6.0, 100 mm NaOAc with ~1000units EndoH (NEB) per 1mg glycosylated protein. Deglycosylation using EndoH leaves a single GlcNAc moiety. Following deglycosylation, proteins were repurified by gel filtration (HiLoad 16/60 Superdex 200; GE Healthcare). IGF2R-Dom11-12 and IGF2R-Dom11-14 proteins were deglycosylated before crystallization whereas the IGF2R-Dom protein used in complex crystallization was not. Electron density consistent with N-linked glycosylation was observed at three of the possible four sites with an Asn-X-Ser/Thr motif; Asn1656 (domain 12), Asn1757 (domain 12) and Asn1816 (domain 13) but not Asn2085 (domain 14). The crystallographic evidence for glycosylation was common to all three structures containing domain 12 and to both structures containing domain 13. 1

2 Details of structure determination and refinement All X-ray diffraction data were collected at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) and were processed using either the HKL (Otwinowski and Minor, 1997) or XDS (Kabsch, 1993) program packages. Crystallographic refinement used CNS (Brünger et al, 1998) and REFMAC5 (Murshudov et al, 1997), with O (Jones et al, 1991) and Coot (Emsley and Cowtan, 2004) used for manual checking and building with reference to 2Fo-Fc and Fo-Fc electron density maps. Ion binding sites were identified from strong positive peaks in Fo-Fc electron density maps. The IGF2R-Dom11-12 structure was solved by selenomethionine phasing using data collected at BM14 (ESRF; Following processing, hkl2map (Pape and Schneider, 2004) was used to locate four selenium sites and produce a map with correlation coefficients CC/CC weak of 58.8%/34.8%. Further refinement of the selenium sites used SOLVE/RESOLVE (Terwilliger, 2000; Terwilliger and Berendzen, 1999). Measurement of the spacing between selenium sites allowed placement of the IGF2R-Dom11 structure (PDB Code 1GP0) into the RESOLVE map. Domain 12 was built manually using domain 11 as a guide, with CALPHA (Esnouf, 1997) used to correct geometry, improve the structure and add side chains. Positional and overall B-factor refinement were performed using REFMAC5. The refined structure comprises residues ; 8 N-terminal residues of the expressed molecule are disordered, as are 3 C- terminal residues plus the His-tag. The electron density is not continuous and the structure is consequently missing residues of the domain 12 AB loop. Where side chain electron density was absent or poor, Ala was substituted for the correct amino acid (Glu1544, Tyr1676, Arg1768, Lys1776, Arg1779, Glu1782, Glu1788, Thr1791, Val1793 and Val1794). The IGF2R-Dom11-12 structure and a poly-ala model based on the main chain of domain 11 were used as molecular replacement models to phase the IGF2R-Dom11-14 data (collected at ESRF ID14-EH3). After an initial solution using the IGF2R-Dom11-12 structure, the poly-ala model was used to locate domains 13 and 14. Several rounds of model building and refinement allowed assignment of side-chains and linkers. Unmodelled electron density was visible where the 2

3 FNII insert was expected to be. An homology model of FNII generated using the CaspR webserver ( was docked into this density and fitted by rigid-body refinement. Positional and overall B-factor refinement were performed using REFMAC5 as above. The refined structure comprises residues ; no electron density is visible for the N- terminus ( ) or for the His-tag and the electron density is not continuous for loop residues and Where side chain electron density was absent or poor, Ala was substituted for the correct amino acid (Thr1686, Asn1687 and Asp1753). The complex data, collected at ESRF ID14-EH2, were also phased by molecular replacement using domains from the IGF2R-Dom11-14 structure. Two molecules were found in the asymmetric unit and unmodelled electron density was visible at the proposed IGF-II binding site for both copies of IGF2R-Dom A further molecular replacement using a crystallographic model for IGF-I (PDB Code 1IMX) placed IGF-I molecules in similar positions relative to each IGF2R fragment, giving two IGF2R-Dom11-13/IGF-II complexes per asymmetric unit (corresponding to a relatively high crystal solvent content of 68%). The models were rigid body refined and the side chains were mutated to the correct IGF-II side chains. Further rounds of positional refinement used CNS (with NCS restraints) and then REFMAC5. Refinement strategy at each stage was validated by a reduction in R free. Final stages of refinement used a recently reported normal-mode-based protocol for modelling anisotropic thermal motions in moderate resolution X- ray structures (Poon et al, 2007). Briefly, REFMAC5 was used to generate TLS parameters (Schomaker and Trueblood, 1968) (one rigid body comprising all protein atoms in the asymmetric unit) and TLSANL (Howlin et al, 1993) used these parameters to include the TLS contributions as ANISOU records in a new PDB file. Programs ENMCALC and OPUSREF (kindly supplied by B.K. Poon and J. Ma, Rice University) were then used to perform an elastic normal mode analysis to produce eigenvectors without the tip effect and to refine the structure using 25 low-frequency normal modes to model anisotropy (Poon et al, 2007). Finally, REFMAC5 was used to refine Cartesian coordinates and overall B-factors with tight restraints on main chain geometry. The 3

4 refined complex structure comprises IGF2R residues and IGF-II residues The rmsd on Cα atoms between the two copies in the asymmetric unit was 0.48 Å for the receptor and 0.12 Å for IGF-II. Residues missing due to absent or poor electron density are the N-terminus ( ), C-terminus ( plus the His-tag) and loop residues and of IGF2R and residues 1-5, 30-39, and of IGF-II. As with the IGF2R-Dom11-14 model, some amino acids (Thr1686, Asn1687 and Asp1753) are substituted with Ala where side chain electron density was absent or poor. The final structures were checked using PROCHECK (Laskowski et al, 1993), which showed no residues in disallowed regions of the Ramachandran Plot, and by WHAT_CHECK (Hooft et al, 1996). Final refinement statistics are given in Table I. 4

5 Supplementary References Brünger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D 54: Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D 60: Esnouf RM (1997) Polyalanine reconstruction from Calpha positions using the program CALPHA can aid initial phasing of data by molecular replacement procedures. Acta Crystallogr D 53: Hooft RWW, Vriend G, Sander C, Abola EE (1996) Errors in protein structures. Nature 381: Howlin B, Butler SA, Moss DS, Harris GW, Driessen HPC (1993) Tlsanl - Tls Parameter-Analysis Program for Segmented Anisotropic Refinement of Macromolecular Structures. J Appl Cryst 26: Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved methods for building protein models in electron- density maps and the location of errors in these models. Acta Crystallogr A 47: Kabsch W (1993) Automatic Processing of Rotation Diffraction Data from Crystals of Initially Unknown Symmetry and Cell Constants. J Appl Cryst 26:

6 Laskowski RA, Macarthur MW, Moss DS, Thornton JM (1993) Procheck - a program to check the stereochemical quality of protein structures. J Appl Cryst 26: Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D 53: Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. In Macromolecular Crystallography, Pt A Vol. 276, pp San Diego: Academic Press Inc Pape T, Schneider TR (2004) HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs. J Appl Cryst 37: Poon BK, Chen X, Lu M, Vyas NK, Quiocho FA, Wang Q, Ma J (2007) Normal mode refinement of anisotropic thermal parameters for a supramolecular complex at 3.42-A crystallographic resolution. Proc Natl Acad Sci USA 104: Schomaker V, Trueblood KN (1968) On Rigid-Body Motion of Molecules in Crystals. Acta Crystallogr B 24: Stanley P (1989) Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity. Mol Cell Biol 9: Terwilliger TC (2000) Maximum-likelihood density modification. Acta Crystallogr D 56: Terwilliger TC, Berendzen J (1999) Automated MAD and MIR structure solution. Acta Crystallogr D 55:

7 Supplementary Table SI Crystallization Details IGF2R-Dom11-12 Protein deglycosylated selenomethionine-labelled Reservoir 0.2 M Potassium sodium tartrate, 20% (w/v) PEG 3350 Cryoprotection reservoir supplemented with 20% (v/v) glycerol Space group P Number of molecules in asymmetric unit 1 Solvent content 51% IGF2R-Dom11-14 Protein deglycolsylated native Reservoir 0.1 M MES ph6.4, 2.5% (w/v) PEG 3000 Cryoprotection reservoir supplemented with 20% (v/v) glycerol Space group C2 Number of molecules in asymmetric unit 1 Solvent content 63% IGF2R-Dom11-13/IGF-II Protein Man 5 GlcNAc 2 glycosylated native Reservoir 0.1 M Tris-HCl ph 9.0, 15% (v/v) MPD Cryoprotection stepwise increase to 30% (v/v) MPD Space group C2 Number of molecules in asymmetric unit 2 Solvent content 68% 7

8 Supplementary Table SII The root-mean-squared deviation (Å) between human IGF2R domains 11, 12, 13 and 14 (: PDB Code 2V5O), bovine IGF2R domains 1, 2 and 3 (bigf2r: PDB Code 1Q25) and bovine CDMPR (bcdmpr: PDB Code 1M6P). Cα equivalences are shown in parentheses. Mean r.m.s.d. between IGF2R domains: Mean r.m.s.d. between IGF2R domains and CDMPR: 2.26 Å (125 C equivalents) 2.38 Å (123 C equivalents) bigf2r Domain 1 bigf2r Domain 2 bigf2r Domain 3 Domain 11 Domain 12 Domain 13 Domain 14 bcdmpr 2.35 (116) 2.12 (115) 2.89 (127) 2.19 (109) 2.21 (124) 2.46 (123) 2.50 (113) 2.41 (135) 2.42 (127) 2.03 (127) 2.14 (117) 2.81 (132) 2.16 (131) 2.11 (129) 2.10 (130) 2.01 (118) 2.44 (131) 2.06 (127) 1.80 (123) 2.32 (130) 1.96 (131) 2.28 (114) 3.26 (121) 2.36 (121) 2.97 (119) 3.07 (118) 2.74 (123) 2.55 (122) bigf2r Domain 1 bigf2r Domain 2 bigf2r Domain 3 Domain 11 Domain 12 Domain 13 Domain 14 bcdmpr Footnote: domains 13 and 14 were built using a single domain polyalanine model based on the main chain structure of domain 11 which may bias the reported r.m.s.d. values. 8

9 Supplementary Figure S1 Sequence alignments and topology information. (A) A structure-based sequence alignment of domains of human IGF2R alongside domains 1-3 of bovine IGF2R. Secondary structure elements are indicated above the alignments, with amino acids forming β-stands on a blue background. Cysteine residues are coloured red and fully conserved residues are in bold. The yellow-boxed PH indicates the insertion point of the FNII domain. (B) Sequence, secondary structure elements and topology of the FNII insert of domain 13. (C) Sequence alignment of IGF-II, IGF-I and insulin. Secondary structure assignments for IGF-II are indicated above the alignments (the yellow bar indicates the flexible loop region). Residues conserved between IGF-II and IGF-I are on a pink background and are also on a pink background if conserved in insulin. The alignment is divided into B, C, A and D domains based on homology with insulin. 9

10 Supplementary Figure S2 Electron density maps. The refined models are shown as a C trace, with domain colors as in Figure 1. Sugars are shown in stick representation. (A) Experimental electron density map for IGF2R-Dom11-12, produced by SOLVE/RESOLVE (Terwilliger, 2000; Terwilliger & Berendzen, 1999) and contoured at 1.5σ. (B) Annealed omit map for the IGF2R-Dom11-13/IGF-II complex produced using CNS (Brünger et al, 1998). A spherical region around Phe19 was omitted and the map is contoured at 1σ. Phe19 is indicated by *. 10

11 Supplementary Figure S3 Change in accessible surface area between IGF2R domain 11, IGF2R FNII and IGF-II upon complexation. Sequences and secondary structure assignments are shown along the x-axes. 11

12 12

13 Supplementary Figure S4 Conformational variation in IGF2R. (A) Loop flexibility in IGF2R domain 11 from four X-ray crystal structures (PDB Codes 1GP0, 2V5N, 2V5O and 2V5P), visualized using the program ESCET with thresholds of between 5 and 10 σ. Regions colored blue are conformationally invariant whereas those colored red show significant differences between the structures. (B) Domain 11 rigid-body shift in IGF2R-domains from uncomplexed to complexed with IGF-II. Cartoon representations of domains from the IGF-II/IGF2R-Dom11-13 complex (blue) superposed onto domains from uncomplexed IGF2R-Dom11-14 (red). For orientation purposes, views of the complex are included. Domain 11 loops involved in IGF-II binding are labelled in the close-up view. 13

14 Supplementary Figure S5 Investigation into IGF2R fragment dimerization using analytical ultracentrifugation. (A) Sedimentation velocity experiments analysis using Gaussian fits. The 2-domain fragment sample, IGF2R-Dom11-12 was at a concentration of 6 mg/ml and the 3-domain fragment, IGF2R- Dom11-13 was 7 mg/ml. (B) c(s,f r ) distributions (see text) for IGF2R-Dom11-12 and for IGF2R-Dom The upper plot for each construct shows a composite of c(s) (a population measure of s, linear function) and c(f r ) (where f r is the frictional ratio with the temperature of the plot (cold blue to hot red) indicating the population of species). The lower panels show s-m plots of s versus weight, M w, using a similar blue-to-red scale A B 14