Surface-exposed Amino Acid Residues Research Articles

Surface-exposed Amino Acid Residues of HPV16 L1 Protein Mediating Interaction with Cell Surface Heparan Sulfate

Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.

Molecular Mechanism of the Reduction of Cysteine Sulfinic Acid of Peroxiredoxin to Cysteine by Mammalian Sulfiredoxin

Among many proteins with cysteine sulfinic acid (Cys-SO2H) residues, the sulfinic forms of certain peroxiredoxins (Prxs) are selectively reduced by sulfiredoxin (Srx) in the presence of ATP. All Srx enzymes contain a conserved cysteine residue. To elucidate the mechanism of the Srx-catalyzed reaction, we generated various mutants of Srx and examined their interaction with PrxI, their ATPase activity, and their ability to reduce sulfinic PrxI. Our results suggest that three surface-exposed amino acid residues, corresponding to Arg50, Asp57, and Asp79 of rat Srx, are critical for substrate recognition. The presence of the sulfinic form (but not the reduced form) of PrxI induces the conserved cysteine of Srx to take the gamma-phosphate of ATP and then immediately transfers the phosphate to the sulfinic moiety of PrxI to generate a sulfinic acid phosphoryl ester (Prx-Cys-S(=O)OPO3(2-)). This ester is reductively cleaved by a thiol molecule (RSH) such as GSH, thioredoxin, and dithiothreitol to produce a disulfide-S-monoxide (Prx-Cys-S(=O)-S-R). The disulfide-S-monoxide is further reduced through the oxidation of three thiol equivalents to complete the catalytic cycle and regenerate Prx-Cys-SH.

MOLECULAR MECHANISM OF THE REDUCTION OF CYSTEINE SULFINIC ACID OF PEROXIREDOXIN TO CYSTEINE BY MAMMALIAN SULFIREDOXIN

Among many proteins with cysteine sulfinic acid (Cys–SO2H) residues, the sulfinic forms of certain peroxiredoxins (Prxs) are selectively reduced by sulfiredoxin (Srx) in the presence of ATP. All Srx enzymes contain a conserved cysteine residue. To elucidate the mechanism of the Srx-catalyzed reaction, we generated various mutants of Srx and examined their interaction with PrxI, their ATPase activity, and their ability to reduce sulfinic PrxI. Our results suggest that three surface-exposed amino acid residues, corresponding to Arg50, Asp57, and Asp79 of rat Srx, are critical for substrate recognition. The presence of the sulfinic form (but not the reduced form) of PrxI induces the conserved cysteine of Srx to take the ã-phosphate of ATP and form a thiophosphate intermediate, which then immediately transfers the phosphate to the sulfinic moiety of PrxI to generate a sulfinic acid phosphoryl ester [Prx-Cys–S(=O)OPO3]. This ester is reductively cleaved by a thiol molecule (RSH) to produce a disulfide-S-monoxide [Prx-Cys–S(=O)–S–R]. The disulfide-S-monoxide is further reduced through the oxidation of three thiol equivalents to complete the catalytic cycle and regenerate Prx-Cys–SH.

Key role of glutamic acid for the cytotoxic activity of the cyclotide cycloviolacin O2.

Cyclotides are cyclic plant proteins with potent cytotoxic effects. Here we systematically probed the importance of surface-exposed charged amino acid residues of the cyclotide cycloviolacin O2, using a strategy involving chemical modifications. We show that the single glutamic acid plays a key role for the cytotoxicity: methylation of this residue produced a 48-fold decrease in potency. Virtually no change in potency was observed when masking the single arginine residue using 1,2-cyclohexanedione, while acetylation of the two lysine residues reduced the potency 3-fold. The derivative with modifications at both arginine and lysine residues showed a 7-fold loss of potency. In addition, we show that the activity is dependent on an intact disulfide network and that the short sequences between the six cysteine residues, that is, the backbone loops, are devoid of cytotoxic activity.

Saccharomyces cerevisiae Ste50 Binds the MAPKKK Ste11 Through a Head-to-tail SAM Domain Interaction

In Saccharomyces cerevisiae, signal transduction through pathways governing mating, osmoregulation, and nitrogen starvation depends upon a direct interaction between the sterile alpha motif (SAM) domains of the Ste11 mitogen-activated protein kinase kinase kinase (MAPKKK) and its regulator Ste50. Previously, we solved the NMR structure of the SAM domain from Ste11 and identified two mutants that diminished binding to the Ste50 SAM domain. Building upon the Ste11 study, we present the NMR structure of the monomeric Ste50 SAM domain and a series of mutants bearing substitutions at surface-exposed hydrophobic amino acid residues. The mid-loop (ML) region of Ste11-SAM, defined by helices H3 and H4 and the end-helix (EH) region of Ste50-SAM, defined by helix H5, were sensitive to substitution, indicating that these two surfaces contribute to the high-affinity interaction. The combination of two mutants, Ste11-SAM-L72R and Ste50-SAM-L69R, formed a high-affinity heterodimer unencumbered by competing homotypic interactions that had prevented earlier NMR studies of the wild-type complex. Yeast bearing mutations that prevented the heterotypic Ste11-Ste50 association in vitro presented signaling defects in the mating and high-osmolarity growth pathways.

Adhesion Molecule ICAM-4 Participates in Erythroblastic Island Formation.

Erythroblasts proliferate, differentiate and enucleate within erythroblastic islands, three dimensional structures comprised of developing erythroblasts surrounding a central macrophage. Collective evidence suggests that erythroblastic islands are highly specialized bone marrow subcompartments where adhesion events, in concert with cytokines, play critical roles in regulating erythropoiesis and apoptosis. ICAM-4, a recently characterized member of the immunoglobulin superfamily, is expressed early in erythroid differentiation. This adhesion molecule interacts with multiple integrin binding partners, including alpha4beta1 and alphaV integrins (alphaVbeta1, alphaVbeta3 and alphaVbeta5). Since erythroblasts express alpha4beta1 and ICAM-4 and macrophages exhibit alphaV, ICAM-4 is an attractive candidate for mediating erythroblast-erythroblast and erythroblast-macrophage attachments. A molecular model of ICAM-4 derived from the crystal structure of closely related ICAM-2 presents the extracellular region of ICAM-4 as two Ig-like domains comprised of A,B,C,D,E,F, and G strands. Employing targeted mutagenesis of surface-exposed amino acid residues, we earlier identified a patch or footprint that mediates adhesion to alphaV integrins comprised of three A strand residues and five G strand residues on N-terminal domain 1. To explore whether ICAM-4 attachments are active in erythroblastic islands we first developed a quantitative live cell assay for reforming islands from single cell suspensions of freshly harvested mouse bone marrow. Islands and their cellular components were identified and quantitated by three-color immunofluorescent microscopy employing fluoresceinated erythroid-specific TER119 antibody, macrophage-specific F4/80 antibody and a DNA probe. To determine the amount of variation in number of islands reformed from a single cell suspension of 1 x 105 cells, we counted islands at the beginning and conclusion of experiments on five different mice. The island numbers were very reproducible and equaled 1000 +/− 158. We then tested the effects of two synthetic peptides that we have previously shown block ICAM-4/alphaV adhesion: peptides FWV and ATSR, corresponding to sequences of the A and G strands of ICAM-4 domain 1, respectively. Both peptides caused a marked, concentration dependent decrease in the percentage of islands formed. 2mM ATSR inhibited island formation by 75% while 2mM FWV inhibited island formation by 70%. In marked contrast, a strand D control peptide had minimal to no effect on island formation. Our data strongly suggest that erythroblast ICAM-4 binding to macrophage alphaV is critical for erythroblastic island formation. We postulate that this newly identified receptor-counterreceptor interaction may be important not only for adhesive integrity of the island structure but also for initiating intracellular signaling essential for normal erythroid terminal differentiation.

Two-prong inhibitors for human carbonic anhydrase II.

The enzyme inhibitors are usually designed by taking into consideration the overall dimensions of the enzyme's active site pockets. This conventional approach often fails to produce desirable affinities of inhibitors for their cognate enzymes. To circumvent such constraints, we contemplated enhancing the binding affinities of inhibitors by attaching tether groups, which would interact with the surface exposed amino acid residues. This strategy has been tested for the inhibition of human carbonic anhydrase II. Benzenesulfonamide serves as a weak inhibitor for the enzyme, but when it is conjugated to iminodiacetate-Cu2+ (which interacts with the surface-exposed His residues) via a spacer group, its binding affinity is enhanced by about 2 orders of magnitude. This "two-prong" approach is expected to serve as a general strategy for converting weak inhibitors of enzymes into tight-binding inhibitors.

Partitioning behavior of amino acids in aqueous two-phase systems containing polyethylene glycol and phosphate buffer

The partitioning behavior of four amino acids, cysteine, phenylalanine, methionine, and lysine in 15 aqueous two-phase systems (ATPSs) with different polyethylene glycol (PEG) molecular weights and phosphate buffers has been studied in the present paper. The phase diagrams of the systems are investigated together with the effect of the PEG molecular weight and pH of the phosphate solutions. The composition of these systems and some parameters such as density and refractive index are determined. The influences of salts in ATPSs, side chain structure of the amino acids, pH of ATPSs, and the PEG molecular weight on the distribution ratios of the amino acids have been studied. This work is useful for the purification of amino acids and the separation of some proteins whose main surface exposed amino acid residues are these four amino acids, respectively.

In vivo neutralization of a C2 domain–specific human anti–Factor VIII inhibitor by an anti-idiotypic antibody

AbstractFactor VIII (FVIII) administration elicits specific inhibitory antibodies (Abs) in about 25% of patients with hemophilia A. The majority of such Abs reacts with FVIII C2 domain. mAbBO2C11 is a high-affinity human monoclonal antibody (mAb) directed toward the C2 domain, which is representative of a major class of human FVIII inhibitors. Anti-idiotypic Abs were raised to mAbBO2C11 to establish their neutralizing potential toward inhibitors. One mouse anti-idiotypic mAb, mAb14C12, specifically prevented mAbBO2C11 binding to FVIII C2 domain and fully neutralized mAbBO2C11 functional inhibitory properties. Modeling of the 3-D conformation of mAb14C12 VH and alignment with the 3-D structure of the C2 domain showed putative 31 surface-exposed amino acid residues either identical or homologous to the C2 domain. These included one C2 phospholipid-binding site, Leu2251-Leu2252, but not Met2199-Phe2200. Forty putative contact residues with mAbBO2C11 were identified. mAb14C12 dose-dependently neutralized mAbBO2C11 inhibitory activity in mice with hemophilia A reconstituted with human recombinant FVIII (rFVIII), allowing full expression of FVIII activity. It also neutralized in an immunoprecipitation assay approximately 50% of polyclonal anti-C2 Abs obtained from 3 of 6 unrelated patients. mAb14C12 is the first example of an anti-idiotypic Ab that fully restores FVIII activity in vivo in the presence of an anti-C2 inhibitor. The present results establish the in vitro and in vivo proof of concept for idiotype-mediated neutralization of a major class of FVIII inhibitors.

Mapping of Der p 2 antibody binding epitopes by site directed mutagenesis

Methods rDer p 2 was subjected to site directed mutagenesis at six selected surface positions (K6, K15, H30, E62, H74, K82) distributed over the entire molecular surface. rDer p 2 mutants containing one (n=6), two (n=2), three (n=1), four (n=1) and six (n=1) mutated amino acids were expressed in Pichia pastoris and analyzed by IgE inhibition and monoclonal antibody (mAb) binding experiments. Results The IgE inhibition experiments demonstrated that mutations in position K6, H30 and E62 interfered marginally with IgE binding, whereas mutations at K15, H74 and K82 significantly reduced IgE binding. A cumulative effect was observed for mutations at H74 and K82, and the double mutant reduced IgE to a level comparable to that of the six position mutant. Three out of four mAbs raised against nDer p 2 were affected by the mutations. The single point mutations at K15 and H74 abolished the interaction between two of the mAbs and rDer p 2 mutants. Concomitant binding analysis of the mAbs indicated that at least 2 distinct epitopes could be identified on nDer p 2. Conclusions Site directed mutagenesis of rDer p 2 identified surface exposed amino acid residues, which participate in both IgE and mAb binding epitopes. Other surface exposed amino acid residues of Der p 2 seem to be located in areas not involved in the binding of the investigated antibodies.

Interactions of Yeast Ribosomal Protein rpS14 with RNA

Yeast ribosomal protein S14 (rpS14) binds to two different RNA molecules: (1) helix 23 of 18 S rRNA during its assembly into 40 S ribosomal subunits and (2) a stem-loop structure in RPS14B pre-mRNA to repress expression of the RPS14B gene. We used the three-dimensional structure of Thermus thermophilus ribosomal protein S11, a bacterial homologue of rpS14, as a guide to identify conserved, surface-exposed amino acid residues that are likely to contact RNA. Eight residues that met these criteria were mutated to alanine. Most of these mutations affected interaction of rpS14 with either helix 23 or the RPS14B stem-loop RNA or both. Assembly of 40 S ribosomal subunits and repression of RPS14B were also affected. S11 contains an extended carboxy-terminal domain rich in basic amino acids, which interacts with rRNA. We systematically evaluated the importance of each of the last ten amino acid residues in the basic, carboxy-terminal tail of yeast rpS14 for binding to RNA, by mutating each to alanine. Mutations in nine of these residues decreased binding of rpS14 to one or both of its RNA ligands. In addition, we examined the importance of four structural motifs in helix 23 of 18 S rRNA for binding to rpS14. Mutations that altered either the terminal loop, the G-U base-pair closing the terminal loop, or the internal loop affected binding of rpS14 to helix 23.

Sequence and structure of human rhinoviruses reveal the basis of receptor discrimination.

The sequences of the capsid protein VP1 of all minor receptor group human rhinoviruses were determined. A phylogenetic analysis revealed that minor group HRVs were not more related to each other than to the nine major group HRVs whose sequences are known. Examination of the surface exposed amino acid residues of HRV1A and HRV2, whose X-ray structures are available, and that of three-dimensional models computed for the remaining eight minor group HRVs indicated a pattern of positively charged residues within the region, which, in HRV2, was shown to be the binding site of the very-low-density lipoprotein (VLDL) receptor. A lysine in the HI loop of VP1 (K224 in HRV2) is strictly conserved within the minor group. It lies in the middle of the footprint of a single repeat of the VLDL receptor on HRV2. Major group virus serotypes exhibit mostly negative charges at the corresponding positions and do not bind the negatively charged VLDL receptor, presumably because of charge repulsion.

Aromatic amino acids at the surface of InlB are essential for host cell invasion by Listeria monocytogenes.

The surface protein InlB of the pathogen Listeria monocytogenes promotes invasion of this bacterium into host cells by binding to and activating the receptor tyrosine kinase Met. The curved leucine-rich repeat (LRR) domain of InlB, which is essential for this process, contains a string of five surface-exposed aromatic amino acid residues positioned along its concave face. Here, we show that the replacement of four of these residues (F104, W124, Y170 or Y214) by serine leads to a complete loss of uptake of latex beads coated with InlB', a truncated functional variant of InlB. The mutants correspondingly display severely reduced binding to Met. To abrogate fully invasion of bacteria expressing full-length InlB, exchange of at least four aromatic amino acids is required. We conclude that InlB binds to Met through its concave surface of the LRR domain, and that aromatic amino acids are critical for binding and signalling before invasion.

The surface exposed amino acid residues of monomeric proteins determine the partitioning in aqueous two-phase systems

It is of great interest and importance how different amino acid residues contribute to and affect the properties of a protein surface. Partitioning in aqueous two-phase systems has the potential to be used as a rapid and simple method for studying the surface properties of proteins. The influence on partitioning of the surface exposed amino acid residues of eight structurally determined monomeric proteins has been studied. The proteins were characterized in terms of surface exposed residues with a computer program, Graphical Representation and Analysis of Surface Properties (GRASP), and partitioned in two EO30PO70–dextran aqueous two-phase systems, only differing in polymer concentrations (system I: 6.8% EO30PO70, 7.1% dextran; system II: 9% EO30PO70, 9% dextran). We show for the first time that the partitioning behaviour of different monomeric proteins can be described by the differences in surface exposed amino acid residues. The contribution to the partition coefficient of the residues was found to be best characterized by peptide partitioning in the aqueous two-phase system. Compared to hydrophobicity scales available in the literature, each amino acid contribution is characterized by the slope given by the graph of log K against peptide chain length, for peptides of different length containing only one kind of residue. It was also shown that each amino acid contribution is relative to the total protein surface and the other residues on the surface. Surface hydrophobicity calculations realized for systems I and II gave respectively correlation coefficients of 0.961 and 0.949 for the linear relation between log K and calculated hydrophobicity values. To study the effect on the partition coefficient of different amino acids, they were grouped into classes according to common characteristics: the presence of an aromatic group, a long aliphatic chain or the presence of charge. Using these groups it was possible to confirm that aromatic residues have the strongest effect on the partition coefficient, giving preference to the upper EO30PO70 phase of the system; on the other hand the presence of charged amino acids on the protein surface enhances the partition of the protein to the lower dextran phase. It is also important to note that the sensitivity of the EO30PO70–dextran system for the surface exposed residues was increased by increasing the polymer concentrations. The partition coefficient of a monomeric protein can thus be predicted from its surface exposed amino acid residues and the system can also be used to characterize protein surfaces of monomeric proteins in general.

Intercellular adhesion molecule-4 binds α4β1 and αV-family integrins through novel integrin-binding mechanisms: Presented in part as abstracts at the 40th annual meeting of the American Society of Hematology, Miami, FL, December 1998 (Blood 1998;92:589a) and at the annual meeting of the British Blood Transfusion Society, Edinburgh, United Kingdom, September 1999 (Transfus Med 1999;9:[suppl 1]9).

The LW blood group glycoprotein, ICAM-4, is a member of the intercellular adhesion molecule (ICAM) family expressed in erythroid cells. To begin to address the function of this molecule, ligands for ICAM-4 on hemopoietic and nonhemopoietic cell lines were identified. Peptide inhibition studies suggest that adhesion of cell lines to an ICAM-4-Fc construct is mediated by an LDV-inhibitable integrin on hemopoietic cells and an RGD-inhibitable integrin on nonhemopoietic cells. Antibody inhibition studies identified the hemopoietic integrin as alpha(4)beta(1.) Antibody inhibition studies on alpha(4)beta(1)-negative, nonhemopoietic cell lines suggested that adhesion of these cells is mediated by alpha(V) integrins (notably alpha(V)beta(1) and alpha(V)beta(5)). The structure of ICAM-4 modeled on the crystal structure of ICAM-2 was used to identify surface-exposed amino acid residues for site-directed mutagenesis. Neither an unusual LETS nor an LDV motif in the first domain of ICAM-4 was critical for integrin binding. ICAM-4 is the first ICAM family member shown to be a ligand for integrins other than those of the beta(2) family, and the data suggest that ICAM-4 has a novel integrin-binding site(s). These findings suggest a role for ICAM-4 in normal erythropoiesis and may also be relevant to the adhesive interactions of sickle cells.

Substitutions of surface amino acid residues of cutinase probed by aqueous two-phase partitioning

The surface properties of a protein are often crucial for recognition and interaction with other molecules. Important functional residues can be identified by mutational analysis. There is a need for rapid methods to study protein surfaces and surface changes due to mutations. Partitioning in aqueous two-phase systems has the potential to be used in this respect since protein partitioning depends on the surface properties of the protein. The influence of surface-exposed amino acid residues in protein partitioning has been studied with cutinase variants, which differed in one or several amino acid residues as a result of site-directed mutagenesis. The solvent accessibility of the mutated residues was determined with a computer program, Graphical Representation and Analysis of Surface Properties. The aqueous two-phase system was composed of dextran and a random copolymer of ethylene oxide and propylene oxide. It was shown, for the first time, to what extent surface-exposed amino acid residues influence the partition coefficient in an aqueous two-phase system. The effect on partitioning could be described only taking into account solvent accessibility and type of residue substitution. The results demonstrate that the system can be used to detect conformational changes in mutant proteins since the expected effect on partitioning due to a mutation can be calculated. The aqueous two-phase system used here does indeed provide a rapid and convenient method to study protein surfaces and slight surface changes due to mutations.

Identification of a Binding Site on the Type II Activin Receptor for Activin and Inhibin

Type II activin receptors (ActRII and ActRIIB) are single-transmembrane domain serine/threonine kinase receptors that bind activin to initiate the signaling and cellular responses triggered by this hormone. Inhibin also binds type II activin receptors and antagonizes many activin effects. Here we describe alanine scanning mutagenesis of the ActRII extracellular domain. We identify a cluster of three hydrophobic residues (Phe(42), Trp(60), and Phe(83)) that, when individually mutated to alanine in the context of the full-length receptor, cause the disruption of activin and inhibin binding to ActRII. Each of the alanine-substituted ActRII mutants retaining activin binding maintains the ability to form cross-linked complexes with activin and supports activin cross-linking to the type I activin receptor ALK4. Unlike wild-type ActRII, the three mutants unable to bind activin do not cause an increase in activin signaling when transiently expressed in a corticotroph cell line. Together, our results implicate these residues in forming a critical binding surface on ActRII required for functional interactions with both activin and inhibin. This first identification of a transforming growth factor-beta family member binding site may provide a general basis for characterizing binding sites for other members of the superfamily.

Definition of receptor binding sites on human interleukin-11 by molecular modeling-guided mutagenesis.

Interleukin-11 (IL-11) belongs to the interleukin-6 (IL-6)-type subfamily of long-chain helical cytokines including IL-6, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M, and cardiotrophin-1, which all share the glycoprotein gp130 as a signal transducing receptor component. IL-11 acts on cells expressing gp130 and the IL-11 receptor (IL-11R) alpha-subunit (IL-11Ralpha). The structural epitopes of IL-11 required for the recruitment of the individual receptor subunits have not yet been defined. Based on the structure of CNTF, a three-dimensional model of human IL-11 was built. Using this model, 10 surface exposed amino acid residues of IL-11 were selected for mutagenesis using analogies to the well-characterized receptor recruitment sites of IL-6, CNTF, and LIF. The respective mutants of human IL-11 were expressed as soluble fusion proteins in bacteria. Their biological activities were determined on HepG2 and Ba/F3-130-11alpha cells. Several mutants with substantially decreased bioactivity and one hyperagonistic mutant were identified and further analyzed with regard to recruitment of IL-11Ralpha and gp130. The low-activity mutant I171D still binds IL-11Ralpha but fails to recruit gp130, whereas the hyperagonistic variant R135E more efficiently engages the IL-11R subunits. The low-activity mutants R190E and L194D failed to bind to IL-11Ralpha. These findings reveal a common mechanism of receptor recruitment in the family of IL-6-type cytokines and offer considerable perspectives for the rational design of IL-11 antagonists and hyperagonists.

Assignment of amino acid residues of the AVR9 peptide of Cladosporium fulvum that determine elicitor activity.

The AVR9 peptide of Cladosporium fulvum is an elicitor of the hypersensitive response in tomato plants carrying the Cf-9 resistance gene (MM-Cf9). To determine the structure-activity relationship of the AVR9 peptide, amino acids important for AVR9 elicitor activity were identified by independently substituting each amino acid of AVR9 by alanine. In addition, surface-exposed amino acid residues of AVR9 were substituted by other amino acids. Activity of the mutant Avr9 constructs was studied by expressing the constructs in MM-Cf9 tomato plants, using the potato virus X (PVX) expression system and assessing the severity of necrosis induced by each PVX::Avr9 construct. This allowed direct identification of amino acid residues of AVR9 that are essential for elicitor activity. We identified amino acid substitutions that resulted in AVR9 mutants with higher, similar, or lower elicitor activity compared to the wild-type AVR9 peptide. Some mutants had completely lost elicitor activity. A selection of peptides, representing different categories, was isolated and injected into leaves of MM-Cf9 plants. The necrosis-inducing activity of the isolated peptides correlated well with the necrosis induced by the corresponding PVX::Avr9 derivatives. Based on the necrosis-inducing activity of the mutant AVR9 peptides and the global structure of AVR9, we assigned sites in AVR9 that are important for its necrosis-inducing activity. We postulate that the "hydrophobic beta-loop" region of the AVR9 peptide is crucial for necrosis-inducing activity in tomato plants that carry the Cf-9 resistance gene.