title: Comparative analyses of the lysine binding site properties of apolipoprotein(a) kringle IV types 7 and 10.
abstract: Apolipoprotein(a) [apo(a)] shares extensive sequence similarity with plasminogen and consists of multiple tandem repeats of domains similar to plasminogen kringle IV (KIV), followed by domains homologous to the plasminogen KV and protease domains. The apo(a) KIV domains can be classified into 10 types on the basis of amino acid sequence (KIV(1)-KIV(10)) of which KIV(10) contains a canonical lysine binding site (LBS); KIV(10) mediates the lysine-dependent interaction of Lp(a) with certain biological substrates. Molecular modeling studies indicated the presence of weak LBS in each of KIV(5)-KIV(8), and subsequent biochemical studies have revealed contributions of these kringles to lysine-mediated interactions involving apo(a). The present study describes the direct demonstration of a weak LBS within KIV(7), as well as the first characterization of the ligand specificity of an LBS outside that of KIV(10). We have expressed both KIV(7) and KIV(10) from bacterial cells and purified them to homogeneity from cell lysates. Equilibrium binding analyses of the KIV(7) LBS using intrinsic fluorescence revealed an affinity for L-lysine and its analogues approximately 10-fold weaker (K(D) = 230 +/- 42 microM for epsilon-aminocaproic acid) than that of KIV(10) (K(D) = 33 +/- 4 microM for epsilon-aminocaproic acid). Moreover, we demonstrated differences in specificity of the LBS of KIV(7) in comparison with KIV(10) in that KIV(7) preferentially bound L-proline. Both kringles bind 4-aminobutyric acid with similar affinities albeit with apparently different mechanisms. Key Phe(62) --> Tyr and Asp(56) --> Glu substitutions in the KIV(7) LBS result in alterations in the size of the LBS and in the spatial relationship between the cationic and anionic centers in the LBS and thus account for the differences in the binding properties of KIV(7) and KIV(10).
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
authors.forename: Mona N
authors.initials: MN
authors.lastname: Rahman
authors.forename: Lev
authors.initials: L
authors.lastname: Becker
authors.forename: Vitali
authors.initials: V
authors.lastname: Petrounevitch
authors.forename: Bruce C
authors.initials: BC
authors.lastname: Hill
authors.forename: Zongchao
authors.initials: Z
authors.lastname: Jia
authors.forename: Marlys L
authors.initials: ML
authors.lastname: Koschinsky
mesh_terms: D001053:Apolipoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053241:Apoprotein(a); D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D018082:Kringles; D017270:Lipoprotein(a) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation
chemical_list: D001053:Apolipoproteins; D017270:Lipoprotein(a); D053241:Apoprotein(a); D008239:Lysine