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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

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Protein Motifs: EGF Domains

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  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

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