Orientation of heparin-binding sites in native vitronectin. Analyses of ligand binding to the primary glycosaminoglycan-binding site indicate that putative secondary sites are not functional.

A primary heparin-binding site in vitronectin has been localized to a cluster of cationic residues near the C terminus of the protein. More recently, secondary binding sites have been proposed. In order to investigate whether the binding site originally identified on vitronectin functions as an exclusive and independent heparin-binding domain, solution binding methods have been used in combination with NMR and recombinant approaches to evaluate ligand binding to the primary site. Evaluation of the ionic strength dependence of heparin binding to vitronectin according to classical linkage theory indicates that a single ionic bond is prominent. It had been previously shown that chemical modification of vitronectin using an arginine-reactive probe results in a significant reduction in heparin binding (Gibson, A., Baburaj, K., Day, D. E., Verhamme, I. , Shore, J. D., and Peterson, C. B. (1997) J. Biol. Chem. 272, 5112-5121). The label has now been localized to arginine residues within the cyanogen bromide fragment-(341-380) that contains the primary heparin-binding site on vitronectin. One- and two-dimensional NMR on model peptides based on this primary heparin-binding site indicate that an arginine residue participates in the ionic interaction and that other nonionic interactions may be involved in forming a complex with heparin. A recombinant polypeptide corresponding to the C-terminal 129 amino acids of vitronectin exhibits heparin-binding affinity that is comparable to that of full-length vitronectin and is equally effective at neutralizing heparin anticoagulant activity. Results from this broad experimental approach argue that the behavior of the primary site is sufficient to account for the heparin binding activity of vitronectin and support an exposed orientation for the site in the structure of the native protein.

Structure-function analysis of a conserved aromatic cluster in the N-terminal domain of human epidermal growth factor.

The importance of a cluster of conserved aromatic residues of human epidermal growth factor (hEGF) to the receptor binding epitope is suggested by the interaction of His10 and Tyr13 of the A-loop with Tyr22 and Tyr29 of the N-terminal beta-sheet to form a hydrophobic surface on the hEGF protein. Indeed, Tyr13 has previously been shown to contribute a hydrophobic determinant to receptor binding. The roles of His10, Tyr22 and Tyr29 were investigated by structure-function analysis of hEGF mutant analogues containing individual replacements of each residue. Substitutions with aromatic residues or a leucine at position 10 retained receptor affinities and agonist activities similar to wild-type indicating that an aromatic residue is not essential. Variants with polar, charged or aliphatic substitutions altered in size and/or hydrophobicity exhibited reduced binding and agonist activities. 1-Dimensional 1H NMR spectra of high, moderate and low-affinity analogues at position 10 suggested only minor alterations in hEGF native structure. In contrast, a variety of replacements were tolerated at position 22 or 29 indicating that neither aromaticity nor hydrophobicity of Tyr22 and Tyr29 is required for receptor binding. CD spectra of mutant analogues at position 22 or 29 indicated a correlation between loss of receptor affinity and alterations in hEGF structure. The results indicate that similar to Tyr13, His10 of hEGF contributes hydrophobicity to the receptor binding epitope, whereas Tyr22 and Tyr29 do not appear to be directly involved in receptor interactions. The latter conclusion, together with previous studies, suggests that hydrophobic residues on only one face of the N-terminal beta-sheet of hEGF are important in receptor recognition.

The functional importance of Leu15 of human epidermal growth factor in receptor binding and activation.

The biological importance of Leu15 of epidermal growth factor (EGF) is suggested by its conservation through evolution, its critical location in the domain-domain interface of EGF and its close proximity to Arg41, a residue that is crucial for receptor binding and activation. Mutagenesis of Leu15 of human EGF (hEGF) was employed to examine the role of this residue in the ligand-receptor interaction. The relative receptor affinities of the hEGF variants, as determined by radioreceptor competition assays, varied depending on the amino acid substitution. The L15F, L15W and L15V hEGF analogues had receptor affinities 45, 26 and 18% respectively of wild type hEGF. The L15A and L15R analogues displayed receptor affinities of only 2.4 and 1.6% relative to wild type hEGF. No binding of the L15E analogue was detected. The relative agonist activities, as measured by receptor tyrosine kinase stimulation assays, generally followed a similar trend. The L15F, L15W and L15V analogues stimulated the receptor kinase to a level (Vmax) similar to that for wild type hEGF. A striking difference was observed between the L15A and L15R variants; although having similar binding affinities, the L15A mutant activated the receptor to only approximately 5% of the wild type Vmax in contrast to 53% for the L15R mutant. 1H-NMR analysis of the L15R and L15A mutants showed only minor structural alterations that were not sufficient to account for the dramatic losses in binding and agonist activities. The results indicate that both the size and hydrophobicity of the gamma-branched aliphatic side chain of Leu15 of hEGF are important in the formation of a catalytically active ligand-receptor complex.

Oxygenation mechanism of ribulose-bisphosphate carboxylase/oxygenase. Structure and origin of 2-carboxytetritol 1,4-bisphosphate, a novel O2-dependent side product generated by a site-directed mutant.

Site-directed mutagenesis has implicated active-site Lys329 of Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in promoting the reaction of CO2 with the 2,3-enediol of ribulose bisphosphate and in stabilizing carboxylation intermediates [Hartman, F. C., & Lee, E. H. (1989) J. Biol. Chem. 264, 11784-11789; Lorimer, G. H., Chen, Y.-R., & Hartman, F. C. (1993) Biochemistry 32, 9018-9024]. Although the K329A mutant is greatly impaired in carboxylation, it catalyzes formation of the enediol, which is misprocessed to an O2-dependent side product [Harpel, M. R., & Hartman, F. C. (1994) Biochemistry 33, 5553-5561]. We now identify this novel side product as 2-carboxytetritol 1,4-bisphosphate (CTBP) by mass spectrometry, 1H-, 13C-, and 31P-NMR spectroscopy, and periodate oxidation. H2O2 accumulates during formation of CTBP, which we show to be derived from a transient precursor, the dicarbonyl D-glycero-2,3-pentodiulose 1,5-bisphosphate. The isolated dicarbonyl bisphosphate is processed by K329A to CTBP. These results, combined with isotope-labeling studies, suggest that CTBP arises by H2O2 elimination from an improperly stabilized peroxy adduct of the enediol intermediate, followed by rearrangement of the resulting dicarbonyl. Therefore, normal oxygenation, as catalyzed by wild-type Rubisco, is not a spontaneous reaction but must involve stabilization of the peroxy intermediate to mitigate formation of the dicarbonyl bisphosphate and subsequently CTBP. CTBP formation verifies the identity of Rubisco's previously invoked oxygenase intermediate, provides additional mechanistic insight into the oxygenation reaction, and shows that Lys329 promotes oxygenation as well as carboxylation. These results may be relevant to other oxygenases, which also exploit substrate carbanions rather than organic cofactors or transition metals for biological oxygen utilization.