The relationship between the L1 and L2 domains of the insulin and epidermal growth factor receptors and leucine-rich repeat modules.

BACKGROUND: Leucine-rich repeats are one of the more common modules found in proteins. The leucine-rich repeat consensus motif is LxxLxLxxNxLxxLxxLxxLxx- where the first 11-12 residues are highly conserved and the remainder of the repeat can vary in size Leucine-rich repeat proteins have been subdivided into seven subfamilies, none of which include members of the epidermal growth factor receptor or insulin receptor families despite the similarity between the 3D structure of the L domains of the type I insulin-like growth factor receptor and some leucine-rich repeat proteins. RESULTS: Here we have used profile searches and multiple sequence alignments to identify the repeat motif Ixx-LxIxx-Nx-Lxx-Lxx-Lxx-Lxx- in the L1 and L2 domains of the insulin receptor and epidermal growth factor receptors. These analyses were aided by reference to the known three dimensional structures of the insulin-like growth factor type I receptor L domains and two members of the leucine rich repeat family, porcine ribonuclease inhibitor and internalin 1B. Pectate lyase, another beta helix protein, can also be seen to contain the sequence motif and much of the structural features characteristic of leucine-rich repeat proteins, despite the existence of major insertions in some of its repeats. CONCLUSION: Multiple sequence alignments and comparisons of the 3D structures has shown that right-handed beta helix proteins such as pectate lyase and the L domains of members of the insulin receptor and epidermal growth factor receptor families, are members of the leucine-rich repeat superfamily.

Identification of a determinant of epidermal growth factor receptor ligand-binding specificity using a truncated, high-affinity form of the ectodomain.

Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF), and human transforming growth factor alpha (hTGF-alpha) with high affinity despite the significant differences in the amino acid sequences of the ligands and the receptors. In contrast, the chicken EGFR can discriminate between mEGF (and hEGF) and hTGF-alpha and binds the EGFs with approximately 100-fold lower affinity. The regions responsible for this poor binding are known to be Arg(45) in hEGF and the L2 domain in the chicken EGFR. In this study we have produced a truncated form of the hEGFR ectodomain comprising residues 1-501 (sEGFR501), which, unlike the full-length hEGFR ectodomain (residues 1-621, sEGFR621), binds hEGF and hTGF-alpha with high affinity (K(D) = 13-21 and 35-40 nM, respectively). sEGFR501 was a competitive inhibitor of EGF-stimulated mitogenesis, being almost 10-fold more effective than the full-length EGFR ectodomain and three times more potent than the neutralizing anti-EGFR monoclonal antibody Mab528. Analytical ultracentrifugation showed that the primary EGF binding sites on sEGFR501 were saturated at an equimolar ratio of ligand and receptor, leading to the formation of a 2:2 EGF:sEGFR501 dimer complex. We have used sEGFR501 to generate three mutants with single position substitutions at Glu(367), Gly(441), or Glu(472) to Lys, the residue found in the corresponding positions in the chicken EGFR. All three mutants bound hTGF-alpha and were recognized by Mab528. However, mutant Gly(441)Lys showed markedly reduced binding to hEGF, implicating Gly(441), in the L2 domain, as part of the binding site that recognizes Arg(45) of hEGF.

Mutant barley (1-->3,1-->4)-beta-glucan endohydrolases with enhanced thermostability.

The similar three-dimensional structures of barley (1-->3)-beta-glucan endohydrolases and (1-->3,1-->4)-beta-glucan endohydrolases indicate that the enzymes are closely related in evolutionary terms. However, the (1-->3)-beta-glucanases hydrolyze polysaccharides of the type found in fungal cell walls and are members of the pathogenesis-related PR2 group of proteins, while the (1-->3,1-->4)-beta-glucanases function in plant cell wall metabolism. The (1-->3)-beta-glucanases have evolved to be significantly more stable than the (1-->3,1-->4)-beta-glucanases, probably as a consequence of the hostile environments imposed upon the plant by invading microorganisms. In attempts to define the molecular basis for the differences in stability, eight amino acid substitutions were introduced into a barley (1-->3,1-->4)-beta-glucanase using site-directed mutagenesis of a cDNA that encodes the enzyme. The amino acid substitutions chosen were based on structural comparisons of the barley (1-->3)- and (1-->3,1-->4)-beta-glucanases and of other higher plant (1-->3)-beta-glucanases. Three of the resulting mutant enzymes showed increased thermostability compared with the wild-type (1-->3,1-->4)-beta-glucanase. The largest increase in stability was observed when the histidine at position 300 was changed to a proline (mutant H300P), a mutation that was likely to decrease the entropy of the unfolded state of the enzyme. Furthermore, the three amino acid substitutions which increased the thermostability of barley (1-->3,1-->4)-beta-glucanase isoenzyme EII were all located in the COOH-terminal loop of the enzyme. Thus, this loop represents a particularly unstable region of the enzyme and could be involved in the initiation of unfolding of the (1-->3,1-->4)-beta-glucanase at elevated temperatures.

The three dimensional structure of the type I insulin-like growth factor receptor.

Ever since the discovery of insulin and its role in the regulation of glucose metabolism, there has been great interest in the molecule itself, the insulin-like growth factors (IGFs), and their receptors (IR and IGF-R). These receptors form a subfamily of tyrosine kinase receptors which are large, transmembrane proteins consisting of several structural domains. Their ectodomains have a similar arrangement of two homologous domains (L1 and L2) separated by a Cys rich region. The C-terminal half of their ectodomains consists of three fibronectin type 3 repeats, and an insert domain that contains the alpha-beta cleavage site. This review summarises the key developments in the understanding of the structure of this family of receptors and their relation to other multidomain proteins. Data presented will include multiple sequence analyses, single molecule electron microscope images of the IGF-1R, insulin receptor (IR), and IR-Fab complexes, and the three dimensional structure of the first three domains of the IGF-1R determined to 2.6 A resolution by x ray crystallography. The L domains each adopt a compact shape consisting of a single stranded, right handed beta-helix. The Cys rich region is composed of eight disulphide bonded modules, seven of which form a rod shaped domain with modules associated in an unusual manner.

Structure and function of the type 1 insulin-like growth factor receptor.

The type 1 insulin-like growth factor receptor (IGF-1R), a transmembrane tyrosine kinase, is widely expressed across many cell types in foetal and postnatal tissues. Activation of the receptor following binding of the secreted growth factor ligands IGF-1 and IGF-2 elicits a repertoire of cellular responses including proliferation, and the protection of cells from programmed cell death or apoptosis. As a result, signalling through the IGF-1R is the principal pathway responsible for somatic growth in foetal mammals, whereas somatic growth in postnatal animals is achieved through the synergistic interaction of growth hormone and the IGFs. Forced overexpression of the IGF-1R results in the malignant transformation of cultured cells: conversely, downregulation of IGF-1R levels can reverse the transformed phenotype of tumour cells, and may render them sensitive to apoptosis in vivo. Elevated levels of IGF-IR are observed in a variety of human tumour types, whereas epidemiological studies implicate the IGF-1 axis as a predisposing factor in the pathogenesis of human breast and prostate cancer. The IGF-1R has thus emerged as a therapeutic target for the development of antitumour agents. Recent progress towards the elucidation of the three-dimensional structure of the extracellular domain of the IGF-1R represents an opportunity for the rational assembly of small molecule antagonists of receptor function for clinical use.

Characterization of a comparative model of the extracellular domain of the epidermal growth factor receptor.

The Epidermal Growth Factor (EGF) receptor is a tyrosine kinase that mediates the biological effects of ligands such as EGF and transforming growth factor alpha. An understanding of the molecular basis of its action has been hindered by a lack of structural and mutational data on the receptor. We have constructed comparative models of the four extracellular domains of the EGF receptor that are based on the structure of the first three domains of the insulin-like growth factor-1 (IGF-1) receptor. The first and third domains of the EGF receptor, L1 and L2, are right-handed beta helices. The second and fourth domains of the EGF receptor, S1 and S2, consist of the modules held together by disulfide bonds, which, except for the first module of the S1 domain, form rod-like structures. The arrangement of the L1 and S1 domains of the model are similar to that of the first two domains of the IGF-1 receptor, whereas that of the L2 and S2 domains appear to be significantly different. Using the EGF receptor model and limited information from the literature, we have proposed a number of regions that may be involved in the functioning of the receptor. In particular, the faces containing the large beta sheets in the L1 and L2 domains have been suggested to be involved with ligand binding of EGF to its receptor.

Biochemical analysis and crystallisation of Fc gamma RIIa, the low affinity receptor for IgG.

Fc gamma RIIa is one of a family of specific cell surface receptors for immunoglobulin. Fc gamma RIIa, which binds immune complexes of certain IgG isotypes, plays important roles in immune homeostasis. However, the precise characteristics of IgG binding and three-dimensional structure of Fc gamma RIIa have not been reported. This study describes the affinity of the Fc gamma RIIa:IgG interaction as well as biochemical characterisation of recombinant Fc gamma RIIa that has been used to generate high quality crystals. Equilibrium binding analysis of the Fc gamma RII:IgG interaction found, IgG3 binds with an affinity of K(D) = 0.6 microM, as expected. Unlike other Fc gamma R, IgG4 also bound to Fc gamma RIIa, K(D) = 3 microM, clearly establishing Fc gamma RIIa as an IgG4 receptor. Biochemical analysis of mammalian and insect cell derived Fc gamma RIIa established the genuine N-terminus with Q being the first amino acid in the sequence Q, A, A, A, P... extending the N-terminus further than previously thought. Furthermore, both potential N-linked glycosylation sites are occupied. Electrospray ionisation mass spectrometry (ESMS) indicate that the N-glycans of baculovirus derived Fc gamma RIIa are core mannose oligosaccharide side chains. Finally, we describe the first crystallisation of diffraction quality crystals of soluble Fc gamma RIIa. Orthorhombic crystals diffract X-rays beyond 2.1 A resolution in the space group P2(1)2(1)2 with cell dimensions a = 78.8 A, b = 100.5 A, c = 27.8 A. This marks a significant advance towards understanding the three-dimensional structure of Fc gamma RIIa and related FcR proteins that share high amino acid identity with Fc gamma RIIa.

Congenital insulin resistance associated with a conformational alteration in a conserved beta-sheet in the insulin receptor L1 domain.

The hormone binding site of members of the insulin receptor family is contained within a highly conserved extracellular region of the receptor. Recent crystallization of the N-terminal region of the binding site revealed two large domains (L1, L2), each organized as a single-stranded right-handed beta-helix, connected by a rod-shaped cysteine-rich domain. Here, we analyze two new naturally occurring mutations in a single beta-sheet within L1, D59G and L62P, that we previously identified in a young woman with classic congenital insulin resistance (type A). Substitution of D59G, a beta-sheet connecting loop residue, caused decreased hormone binding but did not disrupt overall folding, assembly, or movement to the cell surface. In contrast, replacement of the adjacent residue L62P, which is located within the beta-sheet, and positioned in a hormone binding surface, completely disrupted intracellular folding, oligomerization, and trafficking and resulted in aberrant proteolytic degradation. Immunohistochemistry in combination with biosynthetic studies showed that misfolded receptors were retained in an incorrect cellular location and that they colocalized with the resident endoplasmic reticulum chaperone calnexin. This study, together with other mutagenesis data, shows that formation of beta-sheet elements within the L1 beta-helix are critical for the folding of the entire extracellular domain of the receptor and that the hormone contact site is composed in part by residues in this domain.

Crystal structure of the human leukocyte Fc receptor, Fc gammaRIIa.

Fc gamma receptors bind IgG to initiate cellular responses against pathogens and soluble antigens. We have determined the three-dimensional structure of the extracellular portion of human Fc gammaRIIa to 2.0 A resolution providing a structural basis for the unique functions of the leukocyte FcR family. The receptor is composed of two immunoglobulin domains and arranged to expose the ligand-binding site at one end of domain 2. Using alanine mutants we find that the binding sites for IgG1 and 2 are similar but the relative importance of specific regions on the receptor varies. In crystals, Fc gammaRIIa molecules associate to resemble V(L)V(H) dimers, suggesting that two Fc gammaRIIa molecules could cooperate to bind IgG in an asymmetric manner.

Crystal structure of the first three domains of the type-1 insulin-like growth factor receptor.

The type-1 insulin-like growth-factor receptor (IGF-1R) and insulin receptor (IR) are closely related members of the tyrosine-kinase receptor superfamily. IR is essential for glucose homeostasis, whereas IGF-1R is involved in both normal growth and development and malignant transformation. Homologues of these receptors are found in animals as simple as cnidarians. The epidermal growth-factor receptor (EGFR) family is closely related to the IR family and has significant sequence identity to the extracellular portion we describe here. We now present the structure of the first three domains of IGF-IR (L1-Cys-rich-L2) determined to 2.6 A resolution. The L domains each consist of a single-stranded right-handed beta-helix. The Cys-rich region is composed of eight disulphide-bonded modules, seven of which form a rod-shaped domain with modules associated in an unusual manner. The three domains surround a central space of sufficient size to accommodate a ligand molecule. Although the fragment (residues 1-462) does not bind ligand, many of the determinants responsible for hormone binding and ligand specificity map to this central site. This structure therefore shows how the IR subfamily might interact with their ligands.

Polysaccharide hydrolases in germinated barley and their role in the depolymerization of plant and fungal cell walls.

Cell wall degradation is an important event during endosperm mobilization in the germinated barley grain. A battery of polysaccharide and oligosaccharide hydrolases is required for the complete depolymerization of the arabinoxylans and (1 --> 3,1 --> 4)-beta-glucans which comprise in excess of 90% by weight of these walls. The (1 --> 3,1 --> 4)-beta-glucan endohydrolases release oligosaccharides from their substrate and are probably of central importance for the initial solubilization of the (1 --> 3,1 --> 4)-beta-glucans, but beta-glucan exohydrolases and beta-glucosidases may be important additional enzymes for the conversion of released oligosaccharides to glucose. The latter enzymes have recently been purified from germinated barley and characterized. There is an increasing body of evidence to support the notion that the (1 --> 3,1 --> 4)-beta-glucan endohydrolases from germinated barley evolved from the pathogenesis-related (1 --> 3)-beta-glucanases which are widely distributed in plants and which hydrolyse polysaccharides that are abundant in fungal cell walls. Arabinoxylan depolymerization is also mediated by a family of enzymes, but these are less well characterized. (1 --> 4)-beta-Xylan endohydrolases have been purified and the corresponding cDNAs and genes isolated. While the presence of (1 --> 4)-beta-xylan exohydrolases and alpha-L-arabinofuranosidases has been reported many times, the enzymes have not yet been studied in detail. Here, recent advances in the enzymology and physiology of cell wall degradation in the germinated barley grain are briefly reviewed.

Crystallization of the first three domains of the human insulin-like growth factor-1 receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1-462) comprising the L1-cysteine rich-L2 domains of the human IGF-1R ectodomain has been overexpressed in glycosylation-deficient Lec8 cells and has been affinity-purified via a c-myc tag followed by gel filtration. The fragment was recognized by two anti-IGF-1R monoclonal antibodies, 24-31 and 24-60, but showed no detectable binding of IGF-1 or IGF-2. Isocratic elution of IGF-1R/462 on anion-exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 A resolution with cell dimensions a = 77.0 A, b = 99.5 A, c = 120.1 A, and space group P2(1)2(1)2(1).

Molecular cloning of cDNAs encoding (1-->4)-beta-xylan endohydrolases from the aleurone layer of germinated barley (Hordeum vulgare).

Heteroxylans are major constituents of cell walls in the graminaceous monocotyledons. Degradation of walls in the starchy endosperm of germinated cereal grains is mediated, in part at least, by the action of (1-->4)-beta-xylan endohydrolases (EC 3.2.1.8). Complementary DNAs encoding (1-->4)-beta-xylan endohydrolases from the aleurone layer of germinated barley have been isolated and characterized. Southern blot analyses suggest that the enzymes are derived from a family of 3 or 4 genes, and cDNAs corresponding to two of these genes have been sequenced. The amino acid sequence deduced from one cDNA almost exactly matches the amino acid sequence determined previously from the purified enzyme. This enzyme is designated (1-->4)-beta-xylan endohydrolase isoenzyme X-I. The mature enzyme consists of 395 amino acid residues, has a calculated M(r) of ca. 44600 and an isoelectric point of 6.1, and is likely to adopt an (alpha/beta)8 barrel conformation. The amino acid sequence of the barley (1-->4)-beta-xylan endohydrolase encoded by the other cDNA, which is designated isoenzyme X-II, shows ca. 13% sequence divergence compared with isoenzyme X-I. Both enzymes exhibit sequence and structural similarities with microbial xylanases. Expression of the genes in germinated grain appears to be confined largely to the aleurone layer, and no mRNA transcripts could be detected in young vegetative tissues.

Subsite affinities and disposition of catalytic amino acids in the substrate-binding region of barley 1,3-beta-glucanases. Implications in plant-pathogen interactions.

Oligo-1,3-beta-glucosides with degrees of polymerization of 2-9 were labeled at their reducing terminal residues by catalytic tritiation. These substrates were used in detailed kinetic and thermodynamic analyses to examine substrate binding in 1,3-beta-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes GI, GII, and GIII from young seedlings of barley (Hordeum vulgare). Bond-cleavage frequencies, together with the kinetic parameter kcat/Km, have been calculated as a function of substrate chain length to define the number of subsites that accommodate individual beta-glucosyl residues and to estimate binding energies at each subsite. Each isoenzyme has eight beta-glucosyl-binding subsites. The catalytic amino acids are located between the third and fourth subsite from the nonreducing terminus of the substrate. Negative binding energies in subsites adjacent to the hydrolyzed glycosidic linkage suggest that some substrate distortion may occur in this region during binding and that the resultant strain induced in the substrate might facilitate hydrolytic cleavage. If the 1,3-beta-glucanases exert their function as pathogenesis-related proteins by hydrolyzing the branched or substituted 1,3;1,6-beta-glucans of fungal walls, it is clear that relatively extended regions of the cell wall polysaccharide must fit into the substrate-binding cleft of the enzyme.

A tetrad of ionizable amino acids is important for catalysis in barley beta-glucanases.

Determination of the crystal structures of a 1,3-beta-D-glucanase (E.C. 3.2.1.39) and a 1,3-1,4-beta-D-glucanase (E.C. 3.2.1.73) from barley (Hordeum vulgare) (Varghese, J.N, Garrett, T. P. J., Colman, P. M., Chen, L., Høj, P. B., and Fincher, G. B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2785-2789) showed the spatial positions of the catalytic residues in the substrate-binding clefts of the enzymes and also identified highly conserved neighboring amino acid residues. Site-directed mutagenesis of the 1,3-beta-glucanase has now been used to investigate the importance of these residues. Substitution of glutamine for the catalytic nucleophile Glu231 (mutant E231Q) reduced the specific activity about 20,000-fold. In contrast, substitution of glutamine for the catalytic acid Glu288 (mutant E288Q) had less severe consequences, reducing kcat approximately 350-fold with little effect on Km. Substitution of two neighboring and strictly conserved active site-located residues Glu279 (mutant E279Q) and Lys282 (mutant K282M) led to 240- and 2500-fold reductions of Kcat, respectively, with small increases in Km. Thus, a tetrad of ionizable amino acids is required for efficient catalysis in barley beta-glucanases. The active site-directed inhibitor 2,3-epoxypropyl beta-laminaribioside was soaked into native crystals. Crystallographic refinement revealed all four residues (Glu231, Glu279, Lys282, and Glu288) to be in contact with the bound inhibitor, and the orientation of bound substrate in the active site of the glucanase was deduced.

Three-dimensional structures of two plant beta-glucan endohydrolases with distinct substrate specificities.

The three-dimensional structures of (1-->3)-beta-glucanase (EC 3.2.1.39) isoenzyme GII and (1-->3,1-->4)-beta-glucanase (EC 3.2.1.73) isoenzyme EII from barley have been determined by x-ray crystallography at 2.2- to 2.3-A resolution. The two classes of polysaccharide endohydrolase differ in their substrate specificity and function. Thus, the (1-->3)-beta-glucanases, which are classified amongst the plant "pathogenesis-related proteins," can hydrolyze (1-->3)- and (1-->3,1-->6)-beta-glucans of fungal cell walls and may therefore contribute to plant defense strategies, while the (1-->3,1-->4)-beta-glucanases function in plant cell wall hydrolysis during mobilization of the endosperm in germinating grain or during the growth of vegetative tissues. Both enzymes are alpha/beta-barrel structures. The catalytic amino acid residues are located within deep grooves which extend across the enzymes and which probably bind the substrates. Because the polypeptide backbones of the two enzymes are structurally very similar, the differences in their substrate specificities, and hence their widely divergent functions, have been acquired primarily by amino acid substitutions within the groove.

Refinement and analysis of the structure of the first two domains of human CD4.

The structure of a fragment of human CD4 containing two immunoglobulin (Ig)-like domains has been determined by X-ray crystallography and refined at 2.2 A resolution. The structure determination involved iterative building and simulated-annealing refinement, beginning with a partial model. Comparison of domain 1 with an Ig variable domain shows that CD4 has a long and prominent CDR2-like loop (the C"C" corner) and shortened CC' and FG loops (which mediate dimerization in IgV modules). Comparison of domain 2 with Ig modules and domain 1 shows that it can be described as a truncated Ig V domain, in which strands C" and D are deleted. The intersheet disulfide in domain 2 is absent, and there is an altered packing of the two beta-sheets together with a remodeling of the hydrophobic core. The interface between domains 1 and 2 is a lap joint with an extensive hydrophobic surface. The key features of domain 1 that contribute to the interface are found at corresponding positions in domain 2, leading us to propose that the contact between domains 2 and 3 will resemble the one between domains 1 and 2.

Different length peptides bind to HLA-Aw68 similarly at their ends but bulge out in the middle.

We report here the determination and refinement to 1.9 A resolution by X-ray cryo-crystallography the structure of HLA-Aw68. The averaged image from the collection of bound, endogenous peptides clearly shows the atomic structure at the first three and last two amino acids in the peptides but no connected electron density in between. This suggests that bound peptides, held at both ends, take alternative pathways and could be of different lengths by bulging out in the middle. Peptides eluted from HLA-Aw68 include peptides of 9, 10 and 11 amino acids, a direct indication of the length heterogeneity of tightly bound peptides. Peptide sequencing shows relatively conserved 'anchor' residues at position 2 and the carboxy-terminal residue. Conserved binding sites for the peptide N and C termini at the ends of the class I major histocompatibility complex binding groove are apparently dominant in producing the long half-lives of peptide binding and the peptide-dependent stabilization of the class I molecule's structure.

Positioning of a peptide in the cleft of HLA-A2 by complementing amino acid changes.

Several mutant HLA-A2 molecules have been constructed and expressed in the mutant human B-cell line C1R, which lacks HLA-A and HLA-B antigens, and examined for presentation of a previously defined peptide epitope derived from the influenza matrix protein to appropriate human cytotoxic T-lymphocyte lines. When leucine residue 66 in this matrix peptide containing residues 57-68 (matrix peptide 57-68) was replaced by arginine, the resulting matrix peptide 57-68 R66 was not presented to HLA-A2, but the mutation Y116D (tyrosine to aspartic acid at residue 116) in the floor of the peptide binding cleft near its right end dramatically restored peptide presentation. A similar result was obtained by substitution of ornithine for leucine at residue 66. These data provide strong support for a model in which the peptide is orientated with its amino terminus at the left end of the cleft of HLA-A2 and its carboxyl terminus at the right.

Atomic structure of a fragment of human CD4 containing two immunoglobulin-like domains.

The structure of an N-terminal fragment of CD4 has been determined to 2.4 A resolution. It has two tightly abutting domains connected by a continuous beta strand. Both have the immunoglobulin fold, but domain 2 has a truncated beta barrel and a non-standard disulphide bond. The binding sites for monoclonal antibodies, class II major histocompatibility complex molecules, and human immunodeficiency virus gp120 can be mapped on the molecular surface.