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Correlation between the 1.6 A crystal structure and mutational analysis of keratinocyte growth factor.

ABSTRACT: A comprehensive deletion, mutational, and structural analysis of the native recombinant keratinocyte growth factor (KGF) polypeptide has resulted in the identification of the amino acids responsible for its biological activity. One of these KGF mutants (delta23KGF-R144Q) has biological activity comparable to the native protein, and its crystal structure was determined by the multiple isomorphous replacement plus anomalous scattering method (MIRAS). The structure of KGF reveals that it folds into a beta-trefoil motif similar to other members of fibroblast growth factor (FGF) family whose structures have been resolved. This fold consists of 12 anti-parallel beta-strands in which three pairs of the strands form a six-stranded beta-barrel structure and the other three pairs of beta-strands cap the barrel with hairpin triplets forming a triangular array. KGF has 10 well-defined beta strands, which form five double-stranded anti-parallel beta-sheets. A sixth poorly defined beta-strand pair is in the loop between residues 133 and 144, and is defined by only a single hydrogen bond between the two strands. The KGF mutant has 10 additional ordered amino terminus residues (24-33) compared to the other FGF structures, which are important for biological activity. Based on mutagenesis, thermal stability, and structural data we postulate that residues TRP125, THR126, and His127 predominantly confer receptor binding specificity to KGF. Additionally, residues GLN152, GLN138, and THR42 are implicated in heparin binding. The increased thermal stability of delta23KGF-R144Q can structurally be explained by the additional formation of hydrogen bonds between the GLN side chain and a main-chain carbonyl on an adjoining loop. The correlation of the structure and biochemistry of KGF provides a framework for a rational design of this potentially important human therapeutic.

SUBMITTER: Osslund TD

PROVIDER: S-EPMC2144086 | biostudies-other | 1998 Aug

REPOSITORIES: biostudies-other

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Correlation between the 1.6 A crystal structure and mutational analysis of keratinocyte growth factor.

Osslund T D TD Syed R R Singer E E Hsu E W EW Nybo R R Chen B L BL Harvey T T Arakawa T T Narhi L O LO Chirino A A Morris C F CF

Protein science : a publication of the Protein Society 19980801 8

A comprehensive deletion, mutational, and structural analysis of the native recombinant keratinocyte growth factor (KGF) polypeptide has resulted in the identification of the amino acids responsible for its biological activity. One of these KGF mutants (delta23KGF-R144Q) has biological activity comparable to the native protein, and its crystal structure was determined by the multiple isomorphous replacement plus anomalous scattering method (MIRAS). The structure of KGF reveals that it folds into ...[more]

PMID: 10082365

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Project description:N2O is generated by denitrifying bacteria as a product of NO reduction. In denitrification, N2O is metabolized further by the enzyme N2O reductase (N2OR), a multicopper protein which converts N2O into dinitrogen and water. The structure of N2OR remained unknown until the recent elucidation of the structure of the enzyme isolated from Pseudomonas nautica. In the present paper, we report the crystal structure of a blue form of the enzyme that was purified under aerobic conditions from Paracoccus denitrificans. N2OR is a head-to-tail homodimer stabilized by a multitude of interactions including two calcium sites located at the intermonomeric surface. Each monomer is composed of two domains: a C-terminal cupredoxin domain that carries the dinuclear electron entry site known as Cu(A), and an N-terminal seven-bladed beta-propeller domain which hosts the active-site centre Cu(Z). The electrons are transferred from Cu(A) to Cu(Z) across the subunit interface. Cu(Z) is a tetranuclear copper cluster in which the four copper ions (Cu1 to Cu4) are ligated by seven histidine imidazoles, a hydroxyl or water oxygen and a bridging inorganic sulphide. A bound chloride ion near the Cu(Z) active site shares one of the ligand imidazoles of Cu1. This arrangement probably influences the redox potential of Cu1 so that this copper is stabilized in the cupric state. The treatment of N2OR with H2O2 or cyanide causes the disappearance of the optical band at 640 nm, attributed to the Cu(Z) centre. The crystal structure of the enzyme soaked with H2O2 or cyanide suggests that an average of one copper of the Cu(Z) cluster has been lost. The lowest occupancy is observed for Cu3 and Cu4. A docking experiment suggests that N(2)O binds between Cu1 and Cu4 so that the oxygen of N2O replaces the oxygen ligand of Cu4. Certain ligand imidazoles of Cu1 and Cu2, as well as of Cu4, are located at the dimer interface. Particularly those of Cu2 and Cu4 are parts of a bonding network which couples these coppers to the Cu(A) centre in the neighbouring monomer. This structure may provide an efficient electron transfer path for reduction of the bound N2O.

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Correlation between the 1.6 A crystal structure and mutational analysis of keratinocyte growth factor.

Publications

Correlation between the 1.6 A crystal structure and mutational analysis of keratinocyte growth factor.

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