Synergistic use of synchrotron radiation techniques for biological samples in solution: a case study on protein-ligand recognition by the peroxisomal import receptor Pex5p W. A. Stanley, a A. Sokolova, b A. Brown, c D. T. Clarke, c M. Wilmanns a * and D. I. Svergun a,b a EMBL-Hamburg, c/o DESY, Notkestraße 85, 22603 Hamburg, Germany, b Institute of Crystallography, Russian Academy of Sciences, Moscow 117333, Russia, and c Synchrotron Radiation Department, CCLRC Daresbury Laboratory, Warrington WA4 4AD, UK. E-mail: wilmanns@embl-hamburg.de Circular dichroism spectropolarimetry and X-ray scattering data, obtained using synchrotron radiation, can yield information about the secondary and tertiary structure of proteins in solution. These techniques have been used to analyse the architecture and shape of a complex of two proteins in solution. The crystal structures of two separate proteins, the C-terminal domain of Pex5p and SCP2, are available but their complex has not previously been structurally characterized. Circular dichroism spectropolarimetry indicated that complex formation requires little secondary structure rearrangement. X-ray scattering data fit an elongated irregular ‘shoe’-shaped particle of the complex of the two proteins, with dimensions of the order of 30 A ˚  40 A ˚  90 A ˚ . Comparison with the known crystal structures suggests that this ‘shoe’ shape requires a conformational change of the C-terminus of SCP2 to appropriately locate its peroxisomal targeting signal type-1 recognition motif into the binding pocket of the Pex5p receptor. Implications of the combined use of synchrotron- based circular dichroism spectropolarimetry and X-ray scattering in structural biology and proteomics are discussed. Keywords: SRCD; WAXS; SAXS; Pex5p; SCP2; protein structure; peroxisomal targeting; PTS1. 1. Abbreviations BSA: bovine serum albumin. CD: circular dichroism spectropolarimetry. CSA: (+)-10-camphosulphonic acid. DA: dummy atom. d max : maximum particle dimension. DR: dummy residue. DTT: 1,4-dithio-dl-threitol. PDB: Protein Data Bank. Pex5p: peroxin 5 protein, import receptor for PTS1 peroxisomal matrix proteins. Pex5p(C): peroxin 5 protein C-terminal fragment, residues 315–639. p(r): distance distribution function. PTS1: peroxisomal targeting signal type 1. R g : radius of gyration. s: momentum transfer vector, s =4 sin()/, where 2 is the scat- tering angle and  = 1.5 A ˚ is the incident X-ray wavelength. SAXS: small-angle X-ray scattering. SCP2: sterol carrier protein 2, PTS1 containing protein. SRCD: synchrotron radiation circular dichroism spectropolarimetry. TPR: tetratricopeptide repeat motif. WAXS: wide-angle X-ray scattering. UV: ultraviolet light. VUV: vacuum ultraviolet light. 2. Introduction Molecular biology seeks to understand living cells by examining the structure and function of individual biomolecules in the cell. Often these biomolecules do not act alone but are specifically incorporated into higher-order assemblies, or ‘molecular machines’. Considerable effort is expended on understanding their structure–function rela- tionships, particularly those of protein–protein complexes, at various levels of resolution and fidelity. Synchrotron radiation circular dichroism spectropolarimetry (SRCD) has emerged as a powerful technique for evaluating the secondary structure of proteins in solution (Wallace, 2000; Wallace & Janes, 2001; Wallace et al. , 2003). Possible applications of SRCD exceed those from conventional CD spectropolarimeters, which use low-intensity light sources, thus limiting secondary structure analysis to a narrow spectral range in the far-UV (190–250 nm). Synchrotron sources provide a higher photon flux ( 10 3 –10 5 -fold), allowing high- quality data to be extended to the VUV range ( 150–250 nm) with reduced noise from buffer components. The increased spectral range allows additional polypeptide backbone electron transitions to be monitored, hence allowing less ambiguous assignment of secondary structure. Small-angle X-ray scattering (SAXS) can provide information on the shape of proteins in solution. In this technique, the intensity of scattered X-rays, I(s), is measured as a function of the momentum transfer vector, s =4 sin()/, where 2 is the scattering angle and  is the incident X-ray wavelength, usually  1.5 A ˚ , depending on the beamline used (Svergun & Koch, 2002; Koch et al., 2003). Diverse protein systems have benefited from SAXS analysis, recent examples including a study of the domain structure of the multidomain Bruton tyrosine kinase (Marquez et al., 2003); viral capsid assembly (Soko- lova et al., 2001); protein-RNA distribution in a bacterial 70s ribo- some (Svergun & Nierhaus, 2000); and, in combination with geometric docking simulations, analysis of the purine nucleoside phosphorylase trimer (Filgueira de Azevedo et al. , 2003). Depending on experimental conditions, the method can be extended to higher angles, covering the s range  0.5–2.5 A ˚ 1 , to perform a more detailed analysis of protein structure. While this medium- to wide- angle X-ray scattering (WAXS) can readily be applied to protein samples with a high degree of regularity, e.g. spider silk fibres (Riekel & Vollrath, 2001), protein solutions give considerably weaker WAXS signals; indeed, the s range 0.5–1.0 A ˚ 1 is generally regarded as the wide-angle range for protein solutions. Nonetheless, it has been demonstrated that meaningful information on protein architecture can be obtained from protein solutions (Hirai et al., 2002). In combination, therefore, these techniques, SRCD and SAXS/ WAXS, may be used synergistically to study the conformation of proteins in solution, ranging from the secondary, tertiary and to quaternary structural level. Their combined use could allow for studies of individual proteins or higher-order complexes where other techniques may be either of limited use or not applicable. Addi- tionally, they are well suited to investigate conformational changes that may be triggered by, for example, complex formation. We have chosen a complex of two peroxisomal proteins, Pex5p and SCP2, to test the synergistic potential of these methods. While the binding domains of these two proteins are structurally well characterized, little is known about the structure of their complex, which is essential to understand their function. research papers 490 # 2004 International Union of Crystallography doi:10.1107/S090904950402504X J. Synchrotron Rad. (2004). 11, 490–496