Identification of amino acid residues critical for infection with ecotropic murine leukemia retrovirus.

The murine cationic amino acid transporter is also the receptor for murine ecotropic leukemia retrovirus (MuLV-E). Recently, we have cloned a human gene (H13) homologous to the murine ecotropic retroviral receptor (ERR). Although the human homolog is very similar to murine ERR in sequence (87.6% amino acid identity) and structure (14 transmembrane-spanning domains), the human protein fails to function as a receptor for MuLV-E. To identify amino acid residues critical for MuLV-E infection, we took advantage of this species difference and substituted human H13 and murine ERR amino acid residues. Mouse-human chimeric receptor molecules were generated by taking advantage of using common restriction sites. These studies demonstrated that extracellular domains 3 and/or 4 contain the critical amino acid residues. Oligonucleotide-directed mutagenesis was then used to create 13 individual ERR mutants containing one or two amino acids substitutions or insertions within these two extracellular domains. Substitution of as few as one amino acid residue (Tyr) at position 235 in ERR with the corresponding H13 amino acid residue Pro abrogates the ability to function as a receptor for MuLV-E infection. Conversely, substitution of just two amino acid residues at positions 240 and 242 or 242 and 244 in H13 with the corresponding amino acid residues in ERR endows H13 with the ability to function as the receptor. This observation can be utilized to significantly improve the safety of retrovirus-mediated gene therapy in humans.     

Identification of amino acid residues required for Ras p21 target activation.

The Krev-1 gene has been shown to suppress ras-mediated transformation in vitro. Both ras and Krev-1 proteins have identical effector domains (ras residues 32 to 40), which are required for biological activity and for the interaction of Ras p21 with Ras GTPase-activating protein (GAP). In this study, five amino acid residues flanking the ras effector domain, which are not conserved with the Krev-1 protein, were shown to be required for normal protein-protein interactions and biological activity. The substitution of Krev-1 p21 residues 26, 27, 30, 31, and 45 with the corresponding amino acid residues from Ras p21 resulted in a Krev-1 protein which had ras function in both mammalian and yeast biological assays. Replacement of these residues in Ras p21 with the corresponding Krev-1 p21 amino acids resulted in ras proteins which were impaired biologically or reduced in their affinity for in vitro GAP binding. Evaluation of these mutant ras proteins have implications for Ras p21-GAP interactions in vivo.     

Identification of amino acid residues in CD81 critical for interaction with hepatitis C virus envelope glycoprotein E2

Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).     

Identification of amino acid residues critical for the B cell growth-promoting activity of HIV-1 matrix protein p17 variants

Background

HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships.

Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro.

We have probed the structural organization of the human immunodeficiency virus type 1 integrase protein by limited proteolysis and the functional organization by site-directed mutagenesis of selected amino acid residues. A central region of the protein was relatively resistant to proteolysis. Proteins with altered amino acids in this region, or in the N-terminal part of the protein that includes a putative zinc-binding motif, were purified and assayed for 3' processing, DNA strand transfer, and disintegration activities in vitro. In general, these mutations had parallel effects on 3' processing and DNA strand transfer, suggesting that integrase may utilize a single active site for both reactions. The only proteins that were completely inactive in all three assays contained mutations at conserved amino acids in the central region, suggesting that this part of the protein may be involved in catalysis. In contrast, none of the mutations in the N-terminal region resulted in a protein that was inactive in all three assays, suggesting that this part of integrase may not be essential for catalysis. The disintegration reaction was particularly insensitive to these amino acid substitutions, indicating that some function that is important for 3' processing and DNA strand transfer may be dispensable for disintegration.     

Identification of amino acid residues in HIV-1 Vif critical for binding and exclusion of APOBEC3G/F

To define a region(s) in human immunodeficiency virus type 1 (HIV-1) Vif that involves binding to its target APOBEC3G (A3G), we have generated a series of site-specific proviral vif mutants. Of 30 mutants examined, 15 did not grow at all or grew more poorly than wild-type virus in non-permissive cells. Eight clones with N-terminal mutations located outside of the HCCH motif and BC-box, which are known to be directly crucial for the degradation of A3G, were chosen from these growth-defective mutants and mainly analyzed in detail for functional activity of their mutant Vif proteins. By single-cycle replication and immunoprecipitation/immunoblotting analyses, mutants designated W21A, S32A, W38A, Y40A, and H43A were demonstrated to hardly or poorly bind to and neutralize A3G. Upon transfection, these mutants produced progeny virions containing much more A3G than wild-type clone. Interestingly, while mutants designated E76A and W79A acted normally to inactivate A3G, they were found to exhibit a Vif-defective phenotype against A3F. Another unique mutant designated Y69A incompetent against both of A3G/F was also identified. Our results here have indicated that at least two distinct regions in the N-terminal half of HIV-1 Vif are critical for binding and exclusion of A3G/F.     

Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition

Previous studies have shown that human dihydrofolate reductase (DHFR) acts as an RNA-binding protein, in which it binds to its own mRNA and, in so doing, results in translational repression. In this study, we used RNA gel mobility shift and nitrocellulose filter-binding assays to further investigate the specificity of the interaction between human DHFR protein and human DHFR mRNA. Site-directed mutagenesis was used to identify the critical amino acid residues on DHFR protein required for RNA recognition. Human His-Tag DHFR protein specifically binds to human DHFR mRNA, while unrelated proteins including thymidylate synthase, p53 and glutathione-S-transferase were unable to form a ribonucleoprotein complex with DHFR mRNA. The Cys6 residue is essential for RNA recognition, as mutation at this amino acid with either an alanine (C6A) or serine (C6S) residue almost completely abrogated RNA-binding activity. Neither one of the cysteine mutant proteins was able to repress the in vitro translation of human DHFR mRNA. Mutations at amino acids Ile7, Arg28 and Phe34, significantly reduced RNA-binding activity. An RNA footprinting analysis identified three different RNA sequences, bound to DHFR protein, ranging in size from 16 to 45 nt, while a UV cross-linking analysis isolated an ~16 nt RNA sequence bound to DHFR. These studies begin to identify the critical amino acid residues on human DHFR that mediate RNA binding either through forming direct contact points with RNA or through maintaining the protein in an optimal structure that allows for the critical RNA-binding domain to be accessible.     

Identification of amino acid residues in BK virus VP1 that are critical for viability and growth

BK virus (BKV) is a ubiquitous pathogen that establishes a persistent infection in the urinary tract of 80% of the human population. Like other polyomaviruses, the major capsid protein of BKV, virion protein 1 (VP1), is critical for host cell receptor recognition and for proper virion assembly. BKV uses a carbohydrate complex containing alpha(2,3)-linked sialic acid attached to glycoprotein and glycolipid motifs as a cellular receptor. To determine the amino acids important for BKV binding to the sialic acid portion of the complex, we generated a series of 17 point mutations in VP1 and scored them for viral growth. The first set of mutants behaved identically to wild-type virus, suggesting that these amino acids were not critical for virus propagation. Another group of VP1 mutants rendered the virus nonviable. These mutations failed to protect viral DNA from DNase I digestion, indicating a role for these domains in capsid assembly and/or packaging of DNA. A third group of VP1 mutations packaged DNA similarly to the wild type but failed to propagate. The initial burst size of these mutations was similar to that of the wild type, indicating that there is no defect in the lytic release of the mutated virions. Binding experiments revealed that a subset of the BKV mutants were unable to attach to their host cells. These motifs are likely important for sialic acid recognition. We next mapped these mutations onto a model of BKV VP1 to provide atomic insight into the role of these sites in the binding of sialic acid to VP1.     

Functional dissection of cdc37: characterization of domain structure and amino acid residues critical for protein kinase binding

Hsp90 and its co-chaperone Cdc37 facilitate the folding and activation of numerous protein kinases. In this report, we examine the structure-function relationships that regulate the interaction of Cdc37 with Hsp90 and with an Hsp90-dependent kinase, the heme-regulated eIF2alpha kinase (HRI). Limited proteolysis of native and recombinant Cdc37, in conjunction with MALDI-TOF mass spectrometry analysis of peptide fragments and peptide microsequencing, indicates that Cdc37 is comprised of three discrete domains. The N-terminal domain (residues 1-126) interacts with client HRI molecules. Cdc37's middle domain (residues 128-282) interacts with Hsp90, but does not bind to HRI. The C-terminal domain of Cdc37 (residues 283-378) does not bind Hsp90 or kinase, and no functions were ascribable to this domain. Functional assays did, however, suggest that residues S127-G163 of Cdc37 serve as an interdomain switch that modulates the ability of Cdc37 to sense Hsp90's conformation and thereby mediate Hsp90's regulation of Cdc37's kinase-binding activity. Additionally, scanning alanine mutagenesis identified four amino acid residues at the N-terminus of Cdc37 that are critical for high-affinity binding of Cdc37 to client HRI molecules. One mutation, Cdc37/W7A, also implicated this region as an interpreter of Hsp90's conformation. Results illuminate the specific Cdc37 motifs underlying the allosteric interactions that regulate binding of Hsp90-Cdc37 to immature kinase molecules.     

Identification of residues of the H-ras protein critical for functional interaction with guanine nucleotide exchange factors.

Ras proteins are activated in vivo by guanine nucleotide exchange factors encoded by genes homologous to the CDC25 gene of Saccharomyces cerevisiae. We have taken a combined genetic and biochemical approach to probe the sites on Ras proteins important for interaction with such exchange factors and to further probe the mechanism of CDC25-catalyzed GDP-GTP exchange. Random mutagenesis coupled with genetic selection in S. cerevisiae was used to generate second-site mutations within human H-ras-ala15 which could suppress the ability of the Ala-15 substitution to block CDC25 function. We transferred these second-site suppressor mutations to normal H-ras and oncogenic H-rasVal-12 to test whether they induced a general loss of function or whether they selectively affected CDC25 interaction. Four highly selective mutations were discovered, and they affected the surface-located amino acid residues 62, 63, 67, and 69. Two lines of evidence suggested that these residues may be involved in binding to CDC25: (i) using the yeast two-hybrid system, we demonstrated that these mutants cannot bind CDC25 under conditions where the wild-type H-Ras protein can; (ii) we demonstrated that the binding to H-Ras of monoclonal antibody Y13-259, whose epitope has been mapped to residues 63, 65, 66, 67, 70, and 73, is blocked by the mouse sos1 and yeast CDC25 gene products. We also present evidence that the mechanism by which CDC25 catalyzes exchange is more involved than simply catalyzing the release of bound nucleotide and passively allowing nucleotides to rebind. Most critically, a complex of Ras and CDC25 protein, unlike free Fas protein, possesses significantly greater affinity for GTP than for GDP. Furthermore, the Ras CDC25 complex is more readily dissociated into free subunits by GTP than it is by GDP. Both of these results suggest a function for CDC25 in promoting the selective exchange of GTP for GDP.     

A novel strategy for defining critical amino acid residues involved in protein/glycosaminoglycan interactions

The binding of proteins to glycosaminoglycans (GAGs) is the prerequisite for a large number of cellular processes and regulatory events and is associated to many pathologies. However, progress in the understanding of these mechanisms has been hampered by the lack of simple and comprehensive analytical tools for the identification of the structural attributes involved in protein/saccharide interaction. Characterization of GAG binding motifs on proteins has so far relied on site-directed mutagenesis studies, protein sequence mapping using synthetic peptides, molecular modeling, or structural analysis. Here, we report the development of a novel approach for identifying protein residues involved in the binding to heparin, the archetypal member of the GAG family. This method, which uses native proteins, is based on the formation of cross-linked complexes of the protein of interest with heparin beads, the proteolytic digestion of these complexes, and the subsequent identification of the heparin binding containing peptides by N terminus sequencing. Analysis of the CC chemokine regulated on activation, normal T-cell expressed, and secreted (RANTES), the envelope glycoprotein gC from pseudorabies virus and the laminin-5 alpha 3LG4/5 domain validated the techniques and provided novel information on the heparin binding motifs present within these proteins. Our results highlighted this method as a fast and valuable alternative to existing approaches. Application of this technique should greatly contribute to facilitate the structural study of protein/GAG interactions and the understanding of their biological functions.     

Identification of amino acid residues critical for biological activity in human interleukin-18

Interleukin-18 (IL-18) is a pro-inflammatory cytokine, and IL-18-binding protein (IL-18BP) is a naturally occurring protein that binds IL-18 and neutralizes its biological activities. Computer modeling of human IL-18 identified two charged residues, Glu-42 and Lys-89, which interact with oppositely charged amino acid residues buried in a large hydrophobic pocket of IL-18BP. The cell surface IL-18 receptor alpha chain competes with IL-18BP for IL-18 binding, although the IL-18 receptor alpha chain does not share significant homology to IL-18BP. In the present study, Glu-42 was mutated to Lys and Lys-89 to Glu; Glu-42 and Lys-89 were also deleted separately. The deletion mutants (E42X and K89X) were devoid of biological activity, and the K89E mutant lost 95% of its activity. In contrast, compared with wild-type (WT) IL-18, the E42K mutant exhibited a 2-fold increase in biological activity and required a 4-fold greater concentration of IL-18BP for neutralization. The binding of WT IL-18 and its various mutants to human natural killer cells was evaluated by competition assays. The mutant E42K was more effective than WT IL-18 in inhibiting the binding of (125)I-IL-18 to natural killer cells, whereas the three inactive mutants E42X, K89E, and K89X were unable to compete with (125)I-IL-18 for binding. Similarly, WT IL-18 and the E42K mutant induced degradation of Ikappa-Balpha, whereas the three biologically inactive mutants did not induce degradation. The present study reveals that Glu-42 and Lys-89 are critical amino acid residues for the integrity of IL-18 structure and are important for binding to cell surface receptors, for signal transduction, and for neutralization by IL-18BP.     

Ionic interaction of positive amino acid residues of fungal hydrophobin RolA with acidic amino acid residues of cutinase CutL1

Hydrophobins are amphipathic proteins secreted by filamentous fungi. When the industrial fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate co‐adipate (PBSA), it produces RolA, a hydrophobin, and CutL1, a PBSA‐degrading cutinase. Secreted RolA attaches to the surface of the PBSA particles and recruits CutL1, which then condenses on the particles and stimulates the hydrolysis of PBSA. Here, we identified amino acid residues that are required for the RolA–CutL1 interaction by using site‐directed mutagenesis. We quantitatively analyzed kinetic profiles of the interactions between RolA variants and CutL1 variants by using a quartz crystal microbalance (QCM). The QCM analyses revealed that Asp142, Asp171 and Glu31, located on the hydrophilic molecular surface of CutL1, and His32 and Lys34, located in the N‐terminus of RolA, play crucial roles in the RolA–CutL1 interaction via ionic interactions. RolA immobilized on a QCM electrode strongly interacted with CutL1 (K_D = 6.5 nM); however, RolA with CutL1 variants, or RolA variants with CutL1, showed markedly larger K_D values, particularly in the interaction between the double variant RolA‐H32S/K34S and the triple variant CutL1‐E31S/D142S/D171S (K_D = 78.0 nM). We discuss a molecular prototype model of hydrophobin‐based enzyme recruitment at the solid–water interface.     

Human-murine chimeras of ICAM-1 identify amino acid residues critical for rhinovirus and antibody binding.

Human ICAM-1 is the cellular receptor for the major group of human rhinoviruses (HRVs). Previous studies have suggested that the N-terminal domain of ICAM-1 is critical for binding of the major group rhinoviruses. To further define the residues within domain 1 that are involved in virus binding, we constructed an extensive series of ICAM-1 cDNAs containing single and multiple amino acid residue substitutions. In each case, substitutions involved replacement of the human amino acids with those found in murine ICAM-1 to minimize conformational effects. To facilitate the mutagenesis process, a synthetic gene encompassing the first two domains of ICAM-1 was constructed which incorporated 27 additional restriction sites to allow mutagenesis by oligonucleotide replacement. Each of the new constructs was placed into a Rous sarcoma virus vector and expressed in primary chicken embryo fibroblast cells. Binding assays were performed with six major group HRVs, including one high-affinity binding mutant of HRV-14, and two monoclonal antibodies. Results indicated that different serotypes displayed a range of sensitivities to various amino acid substitutions. Amino acid residues of ICAM-1 showing the greatest effect on virus and antibody binding included Pro-28, Lys-29, Leu-30, Leu-37, Lys-40, Ser-67, and Pro-70.