Reductive methylation of lysine residues in acidic fibroblast growth factor: effect on mitogenic activity and heparin affinity

Harper, J. Wade; Lobb, Roy R.

doi:10.1021/bi00402a027

Cited by 64 publications

(56 citation statements)

References 34 publications

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“…A large number of functional studies (involving chemical modification, point mutations, deletion mutations, homologous substitution mutations, and peptide-binding competition studies) in combination with analytical ultracentrifugation, surface plasmon resonance, and affinity chromatography validate the above structural data. [42][43][44][45][46] Heparin binds a specific cluster of basic residues with positive charge density and comprised mostly from the first b-hairpin and the last two-thirds of the third trefoil-fold subdomain (Fig. 3, lower panel).…”

Section: Discussionmentioning

confidence: 99%

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

2012

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The acquisition of function is often associated with destabilizing mutations, giving rise to the stability-function tradeoff hypothesis. To test whether function is also accommodated at the expense of foldability, fibroblast growth factor-1 (FGF-1) was subjected to a comprehensive u-value analysis at each of the 11 turn regions. FGF-1, a b-trefoil fold, represents an excellent model system with which to evaluate the influence of function on foldability: because of its threefold symmetric structure, analysis of FGF-1 allows for direct comparisons between symmetryrelated regions of the protein that are associated with function to those that are not; thus, a structural basis for regions of foldability can potentially be identified. The resulting u-value distribution of FGF-1 is highly polarized, with the majority of positions described as either foldedlike or denatured-like in the folding transition state. Regions important for folding are shown to be asymmetrically distributed within the protein architecture; furthermore, regions associated with function (i.e., heparin-binding affinity and receptor-binding affinity) are localized to regions of the protein that fold after barrier crossing (late in the folding pathway). These results provide experimental support for the foldability-function tradeoff hypothesis in the evolution of FGF-1. Notably, the results identify the potential for folding redundancy in symmetric protein architecture with important implications for protein evolution and design.

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Section: Discussionmentioning

confidence: 99%

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

2012

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“…62 Harper and Lobb 42 recently reported that reductive methylation of less than three lysines in acidic fibroblast growth factor (FGF) significantly reduced its heparin affinity, growth factor receptor affinity, and ability to stimulate mitogenesis in Balb/C 3T3 fibroblasts. Lys-118 was the primary site of dimethylation, and Lys-118 and -112 were the major sites of monomethylation.…”

Section: Hc-ii Contains the [-X-b-b-b-x-x-b-x-] Consensus Given By mentioning

confidence: 99%

“…34 Based on helical wheel diagrams, 35 acidic FGF shows potential to form a-helix. Chemical modification of lysine-112 and lysine-118 by reductive methylation 42 might possibly destabilize the charge interaction with heparin and hence the helical conformation.…”

Section: Molecular Modeling Of Heparin Bindingmentioning

confidence: 99%

Molecular modeling of protein-glycosaminoglycan interactions.

Cardin¹,

Weintraub²

1989

Arteriosclerosis

1,245

993

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. Predictions were then made as to the heparln-blnding domains In endothellal cell growth factor, purpurln, and antithrombln-lll. Many of the natural sequences conforming to these consensus motifs show prominent amphlpathlc periodicities having both a-hellcal and 0-strand conformations as determined by predictive algorithms and circular dlchroism studies. The heparln-blnding domain of vttronectln was modeled and formed a hydrophlllc pocket that wrapped around and folded over a heparln octasaccharide, yielding a complementary structure. We suggest that these consensus sequence elements form potential nucleation sites for the recognition of potyanions In proteins and may provide a useful guide In identifying heparln-blnding regions In other proteins. 1112 However, the structural heterogeneity of the acidic mucopolysaccharides with respect to size, carbohydrate composition and charge, 13 and the variety of proteins that are known to bind to these molecules have complicated a detailed molecular analysis of protein-GAG interactions. The most studied system, binding of heparin to a specific domain or site on human antithrombin-lll (AT-III), involves a unique carbohydrate structure representing only a fraction of the total heparin. 14 The exact amino acid residues on AT-III or other proteins which comprise their heparin-binding domains have not been fully elucidated.To understand the molecular recognition of heparin by proteins, we recently determined the structure of the heparin-binding regions of apolipoprotein (apo) B-100, the major protein constituent of human plasma low density Received June 10, 1988; revision accepted September 14, 1988. lipoproteins (LDL). The structure of these regions was of particular interest as we hypothesized that they could mediate the interaction of LDL with acidic mucopolysaccharides of arterial tissue. We showed that LDL contains five to seven heparin-binding sites on the lipoprotein surface. 15 The heparin-binding cyanogen bromide peptides of apo B-100 were isolated, and their amino acid sequences were determined. 16 These peptides account for five domains of high positive charge density that we suggest mediate the lipoprotein's interaction with glycosaminoglycans. These same regions were also identified by Weisgraber and Rail. 17 We noticed 16 that these domains in apo B-100 have a sequence similarity to the heparinbinding regions of apo E 1819 and human vitronectin (Vn) 20 with respect to the organization of basic and hydropathic residues. The present study extends our initial observations to other proteins whose functions are modified by heparin. We show that all these proteins contain unique consensus sequences of amino acid residues that are potentially involved in heparin binding. MethodsSecondary structure predictions of Vn peptide conformations were performed by the MSEQ program 21 based on Chou-Fasman algorithms 22 and by the method of Gamier et al. 23 Both methods gave comparable predictions of predominant /3-strand and 0-tum structures, and circular dichroism studies on...

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“…Reductive methylation of lysine residues in aFGF has previously suggested the conserved Lys 125 as having a role in heparin binding (Harper & Lobb, 1988). Sitedirected replacement of Lys 125 with glutamic acid also causes significant reduction in heparin binding (Burgess et al, 1990b).…”

Section: Heparin Bindingmentioning

confidence: 99%

Refinement of the structure of human basic fibroblast growth factor at 1.6 Å resolution and analysis of presumed heparin binding sites by selenate substitution

Eriksson

Cousens

Matthews³

1993

Protein Science

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The three‐dimensional structure of human basic fibroblast growth factor has been refined to a crystallographic residual of 16.1% at 1.6 Å resolution. The structure has a Kunitz‐type fold and is composed of 12 antiparallel β‐strands, 6 of which form a β‐barrel. One bound sulfate ion has been identified in the model, hydrogen bonded to the side chains of Asn 27, Arg 120, and Lys 125. The side chain of Arg 120 has two conformations, both of which permit hydrogen bonds to the sulfate. This sulfate binding site has been suggested as the binding site for heparin (Eriksson, A.E., Cousens, L.S., Weaver, L.H., & Matthews, B.W., 1991, Proc. Natl. Acad. Sci. USA 88, 3441–3445). Two β‐mercaptoethanol (BME) molecules are also included in the model, each forming a disulfide bond to the Sγ atoms of Cys 69 and Cys 92, respectively. The side chain of Cys 92 has two conformations of which only one can bind BME. Therefore the BME molecule is half occupied at this site. The locations of possible sulfate binding sites on the protein were examined by replacing the ammonium sulfate in the crystallization medium with ammonium selenate. Diffraction data were measured to 2.2 Å resolution and the structure refined to an R‐factor of 13.8%. The binding of the more electron‐dense selenate ion was identified at two positions. One position was identical to the sulfate binding site identified previously. The second selenate binding site, which is of lower occupancy, is situated 5.6 Å from the first. This ion is hydrogen bonded by the side chain of Lys 135 and Arg 120. Thus the side chain of Arg 120 binds two selenate ions simultaneously. It is suggested that the observed second selenate binding site should also be considered as a possible binding site for heparin, or that both selenate binding sites might simultaneously contribute to the binding of heparin.

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Reductive methylation of lysine residues in acidic fibroblast growth factor: effect on mitogenic activity and heparin affinity

Cited by 64 publications

References 34 publications

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

Experimental support for the foldability–function tradeoff hypothesis: Segregation of the folding nucleus and functional regions in fibroblast growth factor‐1

Molecular modeling of protein-glycosaminoglycan interactions.

Refinement of the structure of human basic fibroblast growth factor at 1.6 Å resolution and analysis of presumed heparin binding sites by selenate substitution

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