title: Crystallographic studies on structural features that determine the enzymatic specificity and potency of human angiogenin: Thr44, Thr80, and residues 38-41.
abstract: Human angiogenin (Ang) is a potent inducer of blood vessel formation and is a member of the pancreatic ribonuclease superfamily. Its enzymatic activity is unusually weak and biased toward cleavage after cytidine nucleotides. As part of an ongoing investigation into the structural basis of Ang's characteristic activity, we have determined the crystal structures of three Ang variants having novel activity. (i) The structure of T44D-Ang indicates that Asp44 can participate directly in pyrimidine binding and that the intrinsic hydrogen-bonding capability of this residue largely governs the pyrimidine specificity of this variant. Unexpectedly, the mutation also causes the most extensive disruption of the C-terminus seen in any Ang variant thus far. This allows the side chain of Arg101 to penetrate the B(1) site, raising the possibility that it participates in substrate binding as occurs in ribonuclease 4. (ii) The structure of T80A-Ang supports the view that Thr80 plays little role in maintaining the obstructive conformation of the C-terminus and that its participation in a hydrogen bond with Thr44 selectively weakens the interaction between Thr44 and N3 of cytosine. (iii) ARH-II is an angiogenin/RNase A chimera in which residues 38-41 of Ang are replaced with the corresponding residues (38-42) of RNase A. Its structure suggests that the guest segment influences catalysis by subtle means, possibly by reducing the pK(a) of the catalytic lysine. The loss of angiogenic activity is not attributable to disruption of known cell-binding or nuclear translocation sites but may be a consequence of the chimera's enhanced ribonucleolytic activity.
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Holloway
authors.forename: Gayatri B
authors.initials: GB
authors.lastname: Chavali
authors.forename: Michelle C
authors.initials: MC
authors.lastname: Hares
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Baker
authors.forename: Gowtham V
authors.initials: GV
authors.lastname: Subbarao
authors.forename: Robert
authors.initials: R
authors.lastname: Shapiro
authors.forename: K Ravi
authors.initials: KR
authors.lastname: Acharya
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D002417:Cattle; D003460:Crystallization; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D013912:Threonine / Q000737:chemistry* / Q000235:genetics
chemical_list: D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013912:Threonine; D001224:Aspartic Acid; C046366:angiogenin; D012259:Ribonuclease, Pancreatic; D000409:Alanine