Journal of Molecular Biology
Structure, Dynamics and Folding of an Immunoglobulin Domain of the Gelation Factor (ABP-120) from Dictyostelium discoideum
Introduction
Filamins are multidomain proteins with characteristic β-pleated sheet repeats that organise filamentous actin into networks of fibres.1, 2, 3, 4 Their importance in maintaining cell structure and generating movement has attracted a great deal of interest, in particular aimed towards elucidating the domain organisation5, 6, 7, 8, 9, 10, 11, 12 and the influence of mechanical stress upon interactions between binding partners.13, 14, 15, 16 The 120-kDa F-actin cross-linking gelation factor from Dictyostelium discoideum (also known as ABP-120 or ddFLN) is one of the most highly studied filamins.17 Its C-terminal rod segment consists of six immunoglobulin-like (Ig) domains. The structure of the fifth Ig domain of ABP-120 (designated as ddFLN5 hereafter) has been determined by X-ray crystallography when conjugated to ddFLN6 at its C-terminus (designated as ddFLN5–6)6 and with ddFLN4 and ddFLN6 flanking both the N- and C-termini (designated as ddFLN4–6).18 Both constructs have rodlike structures with the same mode of domain–domain interactions and form dimeric structures as a result of an extended intermolecular edge-to-edge double β-sheet structure formed within ddFLN6.5, 6, 18 The addition of ddFLN4 at the N-terminus of ddFLN5 does not significantly alter the structure of ddFLN5 in the crystalline state or perturb the interactions between ddFLN5 and ddFLN6, suggesting the existence of a well-defined domain organisation despite the nature of the elongated repeat structure.18
Recently, we have used ddFLN4–6 as a model system through which to study the process of co-translational folding19 and to address the issue of the contribution of the ribosome to folding events that are coupled to protein translation. Nascent chain folding in the cell is inherently associated with the translation process, and the attachment to the ribosome has long been thought to be likely to modify the free-energy landscape sampled by a newly synthesised polypeptide relative to describing the refolding of the protein when isolated.20, 21, 22 By stalling the translation of ddFLN4 or that of ddFLN5–6, ribosome–nascent chain complexes (RNCs) have been generated using a cell-free expression system, enabling subtle structural changes in the ribosomal tunnel within the large ribosomal subunit to be revealed using cryo-electron microscopy image reconstruction.19
Recently, we have used solution-state nuclear magnetic resonance (NMR) spectroscopy to show that the ddFLN of the nascent chain construct can fold into its native structure on the ribosome when tethered to the peptidyl transferase centre via a highly flexible linker corresponding to the largely disordered N-terminal region of ddFLN623 (the RNC that contains the construct of partially translated and stalled ddFLN5–6 is designated as Ig2-RNC hereafter). Other than our NMR work on ddFLN5 as a ribosome-bound nascent chain, little is known about the structure and dynamics of this domain in solution or about its folding properties as an isolated chain. In the present study, we describe the use of a range of solution-state NMR techniques to extract structural and dynamical information about the folded state of ddFLN5, and in addition, we describe the application of NMR and other biophysical methods to monitor the urea-induced unfolding of the protein. Importantly, while circular dichroism (CD) and fluorescence data suggest that ddFLN5 unfolds in the presence of urea in an apparently cooperative manner, residue-specific analysis of the NMR intensities of folded and unfolded populations reveals the existence of a partially structured folding intermediate. The results therefore provide a range of information that describes the properties of the domain and that is also essential for elucidating the underlying differences between the refolding of an isolated protein in a test tube and the de novo folding of a nascent chain in a cell.
Section snippets
Isolated ddFLN5 folds autonomously in solution
We have shown previously by NMR that ddFLN5 can fold into a native-like conformation as a nascent chain while attached to the ribosome through a largely disordered linker encompassing part of ddFLN6.23 Here, we demonstrate the reproducibility of this finding and show that further optimisation of the experiment can yield an improved 15N–1H correlation spectrum of Ig2-RNC (Fig. 1a). The result shows similar differential broadening of a number of cross peaks to those defined previously and that
Discussion
We have used solution-state NMR spectroscopy to characterise the structure and dynamics of an isolated Ig domain, ddFLN5, taken from the C-terminal rod segment of the gelation factor ABP-120 from D. discoideum. The crystal structure of ddFLN5 has been determined previously as part of two-domain and three-domain constructs in which ddFLN6 is linked to the C-terminus of ddFLN5 in both cases and ddFLN4 to the N-terminus of ddFLN5 in the three-domain case.6, 18 These Ig repeats have relatively
Protein expression and purification
The DNA sequence of ddFLN5–6 (residues 644–857) and isolated ddFLN5 (residues 644–750) of ABP-120 from D. discoideum was extracted by PCR and cloned into plasmids containing the T7 RNA polymerase binding site.6 U-15N- and/or U-13C,15N-labelled ddFLN5–6 and ddFLN5 were overexpressed in minimal media using the Escherichia coli BL21 strain as the expression system with (> 98%) 15NH4Cl (1 g/L) and (> 98%) 13C glucose (2 g/L) for 13C labelling as the sole nitrogen and carbon sources, respectively. The
Acknowledgements
S.-T.D.H. is a recipient of a Netherlands Ramsay and a Human Frontier Long-term Fellowship (LT0798/2005). L.D.C. is a National Health and Medical Research Council C.J. Martin Fellow. J.C. and C.M.D. acknowledge funding from the Wellcome and Leverhulme Trusts. J.C. and P.F. are recipients of a Human Frontier Science Program Young Investigator's Award (RGY67/2007). We thank the staff and acknowledge the use of the Biomolecular NMR Facility, Department of Chemistry, University of Cambridge, the
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