Extended and flexible domain solution structure of the extracellular matrix protein anosmin-1 by X-ray scattering, analytical ultracentrifugation and constrained modelling.
Kallmann's syndrome corresponds to a loss of sense of smell and hypogonadotrophic hypogonadism. Defects in anosmin-1 result in the X-linked inherited form of Kallmann's syndrome. Anosmin-1 is an extracellular matrix protein comprised of an N-terminal, cysteine-rich (Cys-box) domain and a whey acidic protein-like (WAP) domain, followed by four fibronectin type III (FnIII) domains. The solution structures of recombinant proteins containing the first three domains (PIWF1) and all six domains (PIWF4) were determined by X-ray scattering and analytical ultracentrifugation. Guinier analyses showed that PIWF1 and PIWF4 have different radii of gyration (R(G)) values of 3.1 nm and 6.7 nm, respectively, but similar cross-sectional radii of gyration (R(XS)) values of 1.5 nm and 1.9 nm, respectively. Distance distribution functions showed that the maximum lengths of PIWF1 and PIWF4 were 11 nm and 23 nm, respectively. Analytical ultracentrifugation gave sedimentation coefficients of 2.52 S and 3.55 S for PIWF1 and PIWF4, respectively. The interpretation of the scattering data by constrained modelling requires homology models for all six domains in anosmin-1. While models were already available for the WAP and FnIII domains, searches suggested the Cys-box domain may resemble the cysteine-rich region of the insulin-like growth factor receptor. Automated constrained molecular modelling based on joining the anosmin-1 domains with structurally randomised linkers resulted in 10,000 models for anosmin-1. A trial-and-error search showed that about 0.1-1.4% of these models fitted the X-ray data. The best models showed that the three domains and six domains in PIWF1 and PIWF4, respectively, were extended. The inter-domain linkers in anosmin-1 could not all be extended at the same time, and there was evidence for inter-domain flexibility. Models with folded-back domain arrangements do not fit the data. These solution structures account for the known biological function of anosmin-1, in particular its ability to interact with its three macromolecular ligands.
Hu
Youli
Y
Centre of Neuroendocrinology, Department of Medicine, Royal Free and University College School of Medicine, Rowland Hill Street, London NW3 2PF, UK.
Sun
Zhe
Z
Eaton
Julian T
JT
Bouloux
Pierre M G
PM
Perkins
Stephen J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D030562:Databases, Protein; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D005353:Fibronectins / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008894:Milk Proteins / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017451:Receptors, Somatomedin / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D014461:Ultracentrifugation; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D014965:X-Rays
C076266:ANOS1 protein, human; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D008024:Ligands; D008894:Milk Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D017451:Receptors, Somatomedin; D011994:Recombinant Proteins; C501364:fibronectin type III like peptide, human; C024471:whey acidic proteins; D066246:ErbB Receptors; D014568:Urokinase-Type Plasminogen Activator