1 / 17

Protein Motifs: EGF Domains

Protein Motifs: EGF Domains. Alison Skinner RCPath Self Help Course 7 th December 2008. Structure of EGF Domains. 30-40 amino acids stabilised by disulphide connectivity Six conserved cysteine residues forming three disulphide bonds in the conformation a n b n a c b c c n c c

chaz
Download Presentation

Protein Motifs: EGF Domains

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Protein Motifs: EGF Domains Alison Skinner RCPath Self Help Course 7th December 2008

  2. Structure of EGF Domains • 30-40 amino acids stabilised by disulphide connectivity • Six conserved cysteine residues forming three disulphide bonds in the conformation anbnacbccncc • Two β-sheets (major N terminal and minor C terminal)

  3. Structure of EGF Domains

  4. Evolutionary History • Two types of EGF domain containing three disulphide bridges • Human EGF (hEGF) • Complement C1r like (cEGF) • Postulated to have arisen from a four disulphide ancestor • Both hEGF and cEGF have the potential to bind Ca2+ ions when the correct consensus sequence is present

  5. Differences Between hEGF and cEGF • hEGF and cEGF domains are differentiated by the location of cysteine cc • hEGF: cc is located in the β turn of the minor sheet • cEGF: cc is located on the second strand of the minor β sheet • Different forms of post translational modification are specific to the different subtypes

  6. EGF Alignments EGF Core

  7. Proteins Containing EGF Domains • Components of the blood coagulation system: • Factor VII, factor IX, factor X, protein C, protein S, thrombomodulin • Connective tissue proteins: • Fibrillin 1, fibrillin 2, LTBP1-4 • Notch pathway proteins: • Notch1, Jagged1, Delta • Low density lipoprotein receptor

  8. Marfan Syndrome and Related Disorders of Microfibrils

  9. Marfan Syndrome – FBN1 • 43/47 of these EGF domains bind calcium • Calcium binding forms stiff rod-like structures • Most mutations in Marfan patients are missense mutations that disrupt the calcium binding EGF domains • EGF domains less saturated by calcium are more flexable than domains completely saturated by calcium. • The ridigity of fibrillin was shown to be reversibly disrupted by the addition of EDTA. • Calcium binding in fibrillin protects against proteolytic degradation • There is post-translational processing of EGF domains • 14 sites for N linked glycosylation • β-hydroxylation of conserved Asn residues within the calcium binding consensus sequence

  10. Haemophilia B (Factor IX) • There are two EGF domains within factor IX • One Ca binding domain essential for biological activity (EGF1) • One non-Ca binding domain which interacts with factor VIIIa (EGF2) • These act as spacers positioning the active site in the correct position • Disrupting mutations within the EGF domains of factor IX cause haemophilia B • p.N64 is a Ca binding ligand within EGF1 • p.N64K has reduced amidolytic activity suggesting that there is interaction between EGF1 and the serine protease domain • Two mutations at p.Pro55 (p.P55S and p.P55L) within the Ca binding EGF domain have been reported to cause mild haemophilia B (coagulant levels 10-12%)

  11. Factor IX – p.P55S • Coagulant level = 10-12% • Antigen level = 50% (suggestive of epitope disruption) • Marginal change to calcium binding ability • Activation by factor VIIa / Tissue factor and factor IXa was normal • Impared ability to activate factor X

  12. Factor IX p.P55L • Coagulant level = 10-12% • Antigen level = 50% (suggestive of epitope disruption) • Marginal change to calcium binding affinity • Aberrant proteolysis between p.R318-S319 • This cleavage is within the active factor IXa • Substrate hydrolysis was 40-50% of normal levels • Increased degradation of protein

  13. Factor IX

  14. Protein S • Non-enzymatic cofactor of activated protein C (APC) which degrades factor Va and VIIIa • Contains four EGF domains • EGF1 mediates interaction with APC • Removal of EGF2 results in loss of function • Replacement of EGF2 with EGF3 (both Ca binding) results in a similar loss of function • The four EGF domains are not equivalent and do not serve a spacer function

  15. Function of EGF domains • Spacer within protein allowing proper conformation • Ca2+ binding gives ridgidity in protein • Protects against proteolysis • Involved in protein / protein interactions • Involved in interactions between intra-protein domains

  16. Bioinformatic Tools that May be Used • Polymorphism phenotyping (PolyPhen) • Sorting tolerant from intolerant (SIFT) • Conservation alignment programme (ClustalW)

  17. References • Deletion or replacement of the second EGF domain of protein S results in loss of APC cofactor activity. Mille-Baker et al., Blood. 2002 • The molecular genetics of Marfan syndrome and microfibrillinopathies. Robinson et al., J Med Genet. 2000; 37 9-25 • Solution structure of a pair of calcium binding epidermal growth factor like domains: Implications to Marfan syndrome and other genetic disorders. Downing et al., Cell. 1996; 95 597-605 • Functional analysis of the EGF-like domain mutations Pro55Ser and Pro55Leu, which cause mild haemophilia B. Knobe et al., J. Thrombosis and Haemostasis. 2003; 1 782-790 • Evolution of distinct EGF domains with specific functions. Wouters et al. Protein Sci. 2005; 14 1091-1103

More Related