Specificity-determining Residues Research Articles

Dissecting the molecular mechanism of drosophila odorant receptors through activity modeling and comparative analysis

To gain insight into the molecular mechanism of odorant receptors (ORs) in Drosophila species, we developed a Quantitative Structure Activity Relationship (QSAR) model that predicts experimentally measured electrophysiological activities between 24 D. melanogaster ORs and 108 odorants. Although the model is limited by the tested odorants,analyzing the model allowed dissection of specific topological and chemical properties necessary for an odorant to elicit excitatory or inhibitory receptor response. Linear odorants with five to eight nonhydrogen atoms at the main chain and hydrogen-bond acceptor and/or hydrogen-bond donor at its ends were found to stimulate strong excitatory response. A comparative sequence analysis of 90 ORs in 15 orthologous groups identified 15 putative specificity-determining residues (SDRs) and 15 globally conserved residues that we postulate as functionally key residues. Mapping to a model of secondary structure resulted in 14 out of 30 key residues locating to the transmembrane (TM) domains. Twelve residues, including six SDRs and six conserved residues, are located at the extracellular halves of the TM domains. Combining the evidence from the QSAR modeling and the comparative sequence analysis, we hypothesize that the Drosophila ORs accept odorants into a binding pocket located on the extracellular halves of its TM domains. The QSAR modeling suggests that the binding pocket is around 15 A in depth and about 6 A in width. Twelve mainly polar or charged key residues, both SDRs and conserved, are located in this pocket and postulated to distinguish docked odorants via primarily geometry fitting and hydrogen-bond interaction.

Exosite Determinants of Serpin Specificity

Serpins form an enormous superfamily of 40-60-kDa proteins found in almost all types of organisms, including humans. Most are one-use suicide substrate serine and cysteine proteinase inhibitors that have evolved to finely regulate complex proteolytic pathways, such as blood coagulation, fibrinolysis, and inflammation. Despite distinct functions for each serpin, there is much redundancy in the primary specificity-determining residues. However, many serpins exploit additional exosites to generate the exquisite specificity that makes a given serpin effective only when certain other criteria, such as the presence of specific cofactors, are met. With a focus on human serpins, this minireview examines use of exosites by nine serpins in the initial complex-forming phase to modulate primary specificity in either binary serpin-proteinase complexes or ternary complexes that additionally employ a protein or other cofactor. A frequent theme is down-regulation of inhibitory activity unless the exosite(s) are engaged. In addition, the use of exosites by maspin and plasminogen activator inhibitor-1 to indirectly affect proteolytic processes is considered.

Three residues at the interface of factor XI (FXI) monomers augment covalent dimerization of FXI.

Human plasma factor XI is a homodimer, with each monomer comprising a catalytic domain and four homologous 'apple' domains. The monomers bind to each other through non-covalent bonds and through a disulfide bond between Cys321 residues in apple 4 domains. To identify residues essential for dimerization in the FXI monomer interface. Specificity-determining residues in apple 4 domains were sought by sequence alignment of FXI and prekallikrein apple domains in different species. Specific residues identified in apple 4 domains were mutagenized and expressed in baby hamster kidney (BHK) cells for evaluation of their effect on FXI dimerization, analyzed by non-reduced sodium dodecylsulfate polyacrylamide gel electrophoresis and size-exclusion chromatography. Among the 19 residues of the FXI monomer interface, Leu284, Ile290 and Tyr329 were defined as specificity-determining residues. Substitutions of these residues or pairs of residues did not affect FXI synthesis and secretion from transfected BHK cells, but did impair dimerization, despite the presence of cysteine at position 321. The double mutant 284A/290A yielded predominantly a monomer, whereas all other single or double mutants yielded monomers as well as disulfide-bonded dimers. The data suggest that Leu284, Ile290 and Tyr329 in the interface of FXI monomers are essential for forming non-covalently bonded dimers that facilitate formation of a disulfide-bonded stable FXI dimer.

Affinity Maturation of a Humanized Rat Antibody for Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals a High Level of Mutational Plasticity Both Inside and Outside the Complementarity-Determining Regions

Antibodies that neutralize RAGE (receptor for advanced glycation end products)-ligand interactions have potential therapeutic applications in both acute and chronic diseases. We generated XT-M4, a rat anti-RAGE monoclonal antibody that has in vivo efficacy in an acute sepsis model. This antibody was subsequently humanized. To improve the affinity of this antibody for the treatment of chronic indications, we used random and targeted mutagenesis strategies in combination with ribosome and phage-display technologies, respectively, to generate libraries of XT-M4 variants. We identified a panel of single-chain Fv antibody fragments (scFv's) that was improved up to 110-fold in a homogeneous time-resolved fluorescence competition assay against parental XT-M4 immunoglobulin G (IgG). After reformatting to bivalent scFv-Fc fusions and IgGs, we observed similar gains in potency in the same assay. Further analysis of binding kinetics as IgG revealed multiple variants with subnanomolar apparent affinity that was dictated primarily by improvements in the off-rate. All variants also had improved binding to cell surface-expressed human RAGE, and all retained, or had improved, apparent affinity for mouse RAGE. F100bL in V(H) (variable region of the heavy chain) complementarity-determining region 3 (CDR3) was one of a number of key mutations that correlated with affinity improvements and was independently identified by both mutagenesis strategies. Random mutagenesis coupled with ribosome display and high-throughput screening revealed an unexpectedly high level of mutational plasticity across the whole length of the humanized scFv, suggesting greater scope for structural optimization outside of the primary antigen-combining site defined by V(H) CDR3 and V(kappa) CDR3. In summary, our comprehensive mutagenesis approach not only achieved the desired affinity maturation of XT-M4 but also defined multiple mutational hotspots across the antibody sequence, provided an insight into the specificity-determining residues of the antibody paratope, and identified additional sites within the CDR loops where human germ-line amino acids may be introduced without affecting function.

Chimpanzees Use More Varied Receptors and Ligands Than Humans for Inhibitory Killer Cell Ig-Like Receptor Recognition of the MHC-C1 and MHC-C2 Epitopes

Humans and chimpanzees have orthologous MHC class I, but few orthologous killer cell Ig-like receptors (KIR). Most divergent are lineage III KIR, which in humans include the inhibitory KIR2DL1 and 2DL2/3 specific for HLA-C. Six lineage III chimpanzee KIR were identified as candidate inhibitory MHC-C receptors and studied using cytolytic assays, to assess the capacity of a defined KIR to function with a defined MHC class I allotype, and direct binding assays with KIR-Fc fusion proteins. Pt-KIR2DL6 and 2DL8 were demonstrated to be inhibitory C1 receptors with a specificity and specificity-determining residue (lysine 44) like KIR2DL3. Analogously, Pt-KIR2DL7 is like KIR2DL1, an inhibitory C2 receptor having methionine 44. Pt-KIR3DL4 and 3DL5 are unusual lineage III KIR with D0 domains, which are also inhibitory C2 receptors with methionine 44. Removal of D0 from KIR3DL, or its addition to KIR2DL, had no effect on KIR function. Pt-KIR2DL9, a fourth inhibitory C2 receptor, has glutamate 44, a previously uncharacterized specificity-determining residue that is absent from human KIR. Reconstruction of the ancestral hominoid KIR sequence shows it encoded lysine 44, indicating that KIR having methionine 44 and glutamate 44 subsequently evolved by independent point substitutions. Thus, MHC-C2-specific KIR have evolved independently on at least two occasions. None of the six chimpanzee KIR studied resembles KIR2DL2, which interacts strongly with C1 and cross-reacts with C2. Whereas human HLA-B allotypes that have functional C1 epitopes are either rare (HLA-B*73) or geographically localized (HLA-B*46), some 25% of Patr-B allotypes have the C1 epitope and are functional KIR ligands.

Prediction of specificity-determining residues for small-molecule kinase inhibitors

BackgroundDesigning small-molecule kinase inhibitors with desirable selectivity profiles is a major challenge in drug discovery. A high-throughput screen for inhibitors of a given kinase will typically yield many compounds that inhibit more than one kinase. A series of chemical modifications are usually required before a compound exhibits an acceptable selectivity profile. Rationalizing the selectivity profile for a small-molecule inhibitor in terms of the specificity-determining kinase residues for that molecule can be an important step toward the goal of developing selective kinase inhibitors.ResultsHere we describe S-Filter, a method that combines sequence and structural information to predict specificity-determining residues for a small molecule and its kinase selectivity profile. Analysis was performed on seven selective kinase inhibitors where a structural basis for selectivity is known. S-Filter correctly predicts specificity determinants that were described by independent groups. S-Filter also predicts a number of novel specificity determinants that can often be justified by further structural comparison.ConclusionS-Filter is a valuable tool for analyzing kinase selectivity profiles. The method identifies potential specificity determinants that are not readily apparent, and provokes further investigation at the structural level.

SDR: a database of predicted specificity-determining residues in proteins

The specificity-determining residue database (SDR database) presents residue positions where mutations are predicted to have changed protein function in large protein families. Because the database pre-calculates predictions on existing protein sequence alignments, users can quickly find the predictions by selecting the appropriate protein family or searching by protein sequence. Predictions can be used to guide mutagenesis or to gain a better understanding of specificity changes in a protein family. The database is available on the web at http://paradox.harvard.edu/sdr.

Identification of selectivity‐determining residues in cytochrome P450 monooxygenases: A systematic analysis of the substrate recognition site 5

The large and diverse family of cytochrome P450 monooxygenases (CYPs) was systematically analyzed to identify selectivity- and specificity-determining residues in the substrate recognition site 5, which is located in close vicinity to the heme center. A positively charged heme-interacting residue was identified in the structures of 29 monooxygenases and in 97.7% of the 6379 CYP sequences investigated here. This heme-interacting residue restricts the conformation of the substrate recognition site 5 and is preferentially located at position 10 or 11 after the conserved ExxR motif (in 94.4% of the sequences), in 3.3% of the sequences at position 9 or 12. As a result, a classification by the position of the heme-interacting residue allows to predict residues that are closest to the heme center and restrict its accessibility. In 98.4% of all CYP sequences a preferentially hydrophobic residue is located at position 5 after the ExxR motif that is predicted to point close to the heme center. Replacing this residue by hydrophobic residues of different size has been shown to change substrate specificity and regioselectivity for CYPs of different superfamilies. Twenty-seven percent of all CYPs are predicted to contain a second selectivity-determining residue at position 9 after the ExxR motif that can be identified by the pattern EXXR-X(7)-{P}-x-P-[HKR].

Reverse Conservation Analysis Reveals the Specificity Determining Residues of Cytochrome P450 Family 2 (CYP 2)

The concept of conservation of amino acids is widely used to identify important alignment positions of orthologs. The assumption is that important amino acid residues will be conserved in the protein family during the evolutionary process. For paralog alignment, on the other hand, the opposite concept can be used to identify residues that are responsible for specificity. Assuming that the function-specific or ligand-specific residue positions will have higher diversity since they are under evolutionary pressure to fit the target specificity, these function-specific or ligand-specific residues positions will have a lower degree of conservation than other positions in a highly conserved paralog alignment. This study assessed the ability of reverse conservation analysis to identify function-specific and ligand-specific residue positions in closely related paralog. Reverse conservation analysis of paralog alignments successfully identified all six previously reported substrate recognition sites (SRSs) in cytochrome P450 family 2 (CYP 2). Further analysis of each subfamily identified the specificity-determining residues (SDRs) that have been experimentally found. New potential SDRs were also predicted and await confirmation by further experiments or modeling calculations. This concept may be also applied to identify SDRs in other protein families.

Dynamic Scaffolding in a G Protein-Coupled Signaling System

The INAD scaffold organizes a multiprotein complex that is essential for proper visual signaling in Drosophila photoreceptor cells. Here we show that one of the INAD PDZ domains (PDZ5) exists in a redox-dependent equilibrium between two conformations--a reduced form that is similar to the structure of other PDZ domains, and an oxidized form in which the ligand-binding site is distorted through formation of a strong intramolecular disulfide bond. We demonstrate transient light-dependent formation of this disulfide bond in vivo and find that transgenic flies expressing a mutant INAD in which PDZ5 is locked in the reduced state display severe defects in termination of visual responses and visually mediated reflex behavior. These studies demonstrate a conformational switch mechanism for PDZ domain function and suggest that INAD behaves more like a dynamic machine rather than a passive scaffold, regulating signal transduction at the millisecond timescale through cycles of conformational change.

Sequence harmony: detecting functional specificity from alignments

Multiple sequence alignments are often used for the identification of key specificity-determining residues within protein families. We present a web server implementation of the Sequence Harmony (SH) method previously introduced. SH accurately detects subfamily specific positions from a multiple alignment by scoring compositional differences between subfamilies, without imposing conservation. The SH web server allows a quick selection of subtype specific sites from a multiple alignment given a subfamily grouping. In addition, it allows the predicted sites to be directly mapped onto a protein structure and displayed. We demonstrate the use of the SH server using the family of plant mitochondrial alternative oxidases (AOX). In addition, we illustrate the usefulness of combining sequence and structural information by showing that the predicted sites are clustered into a few distinct regions in an AOX homology model. The SH web server can be accessed at www.ibi.vu.nl/programs/seqharmwww.

Local structural disorder imparts plasticity on linear motifs

The dynamic nature of protein interaction networks requires fast and transient molecular switches. The underlying recognition motifs (linear motifs, LMs) are usually short and evolutionarily variable segments, which in several cases, such as phosphorylation sites or SH3-binding regions, fall into locally disordered regions. We probed the generality of this phenomenon by predicting the intrinsic disorder of all LM-containing proteins enlisted in the Eukaryotic Linear Motif (ELM) database. We demonstrated that LMs in average are embedded in locally unstructured regions, while their amino acid composition and charge/hydropathy properties exhibit a mixture characteristic of folded and disordered proteins. Overall, LMs are constructed by grafting a few specificity-determining residues favoring structural order on a highly flexible carrier region. These results establish a connection between LMs and molecular recognition elements of intrinsically unstructured proteins (IUPs), which realize a non-conventional mode of partner binding mostly in regulatory functions.

Structural Descriptors of gp120 V3 Loop for the Prediction of HIV-1 Coreceptor Usage

HIV-1 cell entry commonly uses, in addition to CD4, one of the chemokine receptors CCR5 or CXCR4 as coreceptor. Knowledge of coreceptor usage is critical for monitoring disease progression as well as for supporting therapy with the novel drug class of coreceptor antagonists. Predictive methods for inferring coreceptor usage based on the third hypervariable (V3) loop region of the viral gene coding for the envelope protein gp120 can provide us with these monitoring facilities while avoiding expensive phenotypic tests. All simple heuristics (such as the 11/25 rule) as well as statistical learning methods proposed to date predict coreceptor usage based on sequence features of the V3 loop exclusively. Here, we show, based on a recently resolved structure of gp120 with an untruncated V3 loop, that using structural information on the V3 loop in combination with sequence features of V3 variants improves prediction of coreceptor usage. In particular, we propose a distance-based descriptor of the spatial arrangement of physicochemical properties that increases discriminative performance. For a fixed specificity of 0.95, a sensitivity of 0.77 was achieved, improving further to 0.80 when combined with a sequence-based representation using amino acid indicators. This compares favorably with the sensitivities of 0.62 for the traditional 11/25 rule and 0.73 for a prediction based on sequence information as input to a support vector machine and constitutes a statistically significant improvement. A detailed analysis and interpretation of structural features important for classification shows the relevance of several specific hydrogen-bond donor sites and aliphatic side chains to coreceptor specificity towards CCR5 or CXCR4. Furthermore, an analysis of side chain orientation of the specificity-determining residues suggests a major role of one side of the V3 loop in the selection of the coreceptor. The proposed method constitutes the first approach to an improved prediction of coreceptor usage based on an original integration of structural bioinformatics methods with statistical learning.

Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach

BackgroundThe large amount of available sequence information for the plant acyl-ACP thioesterases (TEs) made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL) program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium.ResultsSubstitutions at four of the positions (74, 86, 141, and 174) changed substrate specificity to varying degrees while changes at the remaining two positions, 110 and 221, essentially inactivated the thioesterase. The effects of substitutions at positions 74, 141, and 174 (3-MUT) or 74, 86, 141, 174 (4-MUT) were not additive with respect to specificity.ConclusionFour of six putative specificity determining positions in plant TEs, identified with the use of CPDL, were validated experimentally; a novel colorimetric screen that discriminates between active and inactive TEs is also presented.

Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF

Mono-, di- and trimethylated states of particular histone lysine residues are selectively found in different regions of chromatin, thereby implying specialized biological functions for these marks ranging from heterochromatin formation to X-chromosome inactivation and transcriptional regulation. A major challenge in chromatin biology has centred on efforts to define the connection between specific methylation states and distinct biological read-outs impacting on function. For example, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with transcription start sites of active genes, but the molecular 'effectors' involved in specific recognition of H3K4me3 tails remain poorly understood. Here we demonstrate the molecular basis for specific recognition of H3(1-15)K4me3 (residues 1-15 of histone H3 trimethylated at K4) by a plant homeodomain (PHD) finger of human BPTF (bromodomain and PHD domain transcription factor), the largest subunit of the ATP-dependent chromatin-remodelling complex, NURF (nucleosome remodelling factor). We report on crystallographic and NMR structures of the bromodomain-proximal PHD finger of BPTF in free and H3(1-15)K4me3-bound states. H3(1-15)K4me3 interacts through anti-parallel beta-sheet formation on the surface of the PHD finger, with the long side chains of arginine 2 (R2) and K4me3 fitting snugly in adjacent pre-formed surface pockets, and bracketing an invariant tryptophan. The observed stapling role by non-adjacent R2 and K4me3 provides a molecular explanation for H3K4me3 site specificity. Binding studies establish that the BPTF PHD finger exhibits a modest preference for K4me3- over K4me2-containing H3 peptides, and discriminates against monomethylated and unmodified counterparts. Furthermore, we identified key specificity-determining residues from binding studies of H3(1-15)K4me3 with PHD finger point mutants. Our findings call attention to the PHD finger as a previously uncharacterized chromatin-binding module found in a large number of chromatin-associated proteins.

Construction, Affinity Maturation, and Biological Characterization of an Anti-tumor-associated Glycoprotein-72 Humanized Antibody

The tumor-associated glycoprotein (TAG)-72 is expressed in the majority of human adenocarcinomas but is rarely expressed in most normal tissues, which makes it a potential target for the diagnosis and therapy of a variety of human cancers. Here we describe the construction, affinity maturation, and biological characterization of an anti-TAG-72 humanized antibody with minimum potential immunogenicity. The humanized antibody was constructed by grafting only the specificity-determining residues (SDRs) within the complementarity-determining regions (CDRs) onto homologous human immunoglobulin germ line segments while retaining two mouse heavy chain framework residues that support the conformation of the CDRs. The resulting humanized antibody (AKA) showed only about 2-fold lower affinity compared with the original murine monoclonal antibody CC49 and 27-fold lower reactivity to patient serum compared with the humanized antibody HuCC49 that was constructed by CDR grafting. The affinity of AKA was improved by random mutagenesis of the heavy chain CDR3 (HCDR3). The highest affinity variant (3E8) showed 22-fold higher affinity compared with AKA and retained the original epitope specificity. Mutational analysis of the HCDR3 residues revealed that the replacement of Asn(97) by isoleucine or valine was critical for the affinity maturation. The 3E8 labeled with (125)I or (131)I showed efficient tumor targeting or therapeutic effects, respectively, in athymic mice with human colon carcinoma xenografts, suggesting that 3E8 may be beneficial for the diagnosis and therapy of tumors expressing TAG-72.

Sequence variation in trypsin‐ and chymotrypsin‐like cDNAs from the midgut of Ostrinia nubilalis: methods for allelic differentiation of candidate Bacillus thuringiensis resistance genes

Midgut expressed alkaline serine proteases of Lepidoptera function in conversion of Bacillus thuringiensis (Bt) protoxin to active toxin, and reduced level of transcript T23 is associated with Ostrinia nubilalis resistance to Dipel Bt formulations. Three groups of trypsin- (OnT25, OnT23, and OnT3) and two chymotrypsin-like (OnC1 and OnC2) cDNAs were isolated from O. nubilalis midgut tissue. Intraspecific groupings are based on cDNA similarity and peptide phylogeny. Derived serine proteases showed a catalytic triad (His, Asp, and Ser; except transcript OnT23a), three substrate specificity-determining residues, and three paired disulphide bonds. RT-PCR indicated all transcripts are expressed in the midgut. Mendelian-inherited genomic markers for loci OnT23, OnT3 and OnC1 will be useful for association of alleles with bioassayed Bt toxin resistance phenotypes.

Design and validation of a synthetic VH repertoire with tailored diversity for protein recognition

Previous studies have indicated differences in the specificity-determining residues (SDRs) of antibodies that recognize haptens, peptides, or proteins. Here, we designed a V(H) repertoire based on the human scaffold 3-23/J(H)4 and diversification of high and medium-usage SDRs of anti-protein and anti-peptide antibodies. The repertoire was synthesized by overlapping polymerase chain reaction (PCR) and combined with the V(L) chain of the anti-hen egg-white lysozyme (HEL) antibody D1.3. The resulting chimeric single-chain Fv fragments (scFvs) phage-displayed library was panned in HEL-coated immunotubes. After two rounds of selection under non-stringent conditions, that is, trypsinization after 2 h of incubation at room temperature, 63 of 167 clones analyzed (38%) were found to express scFvs specific to HEL. Twenty clones were characterized by DNA sequencing resulting in 10 unique scFvs. Interestingly, the panel of unique scFvs was highly diverse, with V(H) sequences differing in 16 of the 17 positions variegated in the repertoire. Thus, diverse chemico-physical and structural solutions were selected from the library, even when the V(H) repertoire was constrained by the V(L) chain of D1.3 to yield binders against a definite region of HEL surface. The more often selected scFvs, namely H6-1 and B7-1, which differed in eight SDRs, showed levels of expression in E. coli TG1 strain, 6 and 10 times higher than the parental D1.3 Fv fragment, respectively. Dissociation constants (K(Ds)) measured in the BIAcore were 11 and 6.6 nM for H6-1 and B7-1, respectively. These values compared well to the K(D) of 4.7 nM measured for D1.3, indicating that the V(H) repertoire here designed is a valuable source of diverse, well-expressed and high affinity V(H) domains.

Structural Basis for Recognition and Sequestration of UUU OH 3′ Temini of Nascent RNA Polymerase III Transcripts by La, a Rheumatic Disease Autoantigen

The nuclear phosphoprotein La was identified as an autoantigen in patients with systemic lupus erythematosus and Sjogren's syndrome. La binds to and protects the UUU(OH) 3' terminii of nascent RNA polymerase III transcripts from exonuclease digestion. We report the 1.85 angstroms crystal structure of the N-terminal domain of human La, consisting of La and RRM1 motifs, bound to r(U1-G2-C3-U4-G5-U6-U7-U8-U9OH). The U7-U8-U9OH 3' end, in a splayed-apart orientation, is sequestered within a basic and aromatic amino acid-lined cleft between the La and RRM1 motifs. The specificity-determining U8 residue bridges both motifs, in part through unprecedented targeting of the beta sheet edge, rather than the anticipated face, of the RRM1 motif. Our structural observations, supported by mutation studies of both La and RNA components, illustrate the principles behind RNA sequestration by a rheumatic disease autoantigen, whereby the UUU(OH) 3' ends of nascent RNA transcripts are protected during downstream processing and maturation events.

An evolution-based analysis scheme to identify CO2/O2 specificity-determining factors for ribulose 1,5-bisphosphate carboxylase/oxygenase

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) catalyzes a rate-limiting step in photosynthetic carbon assimilation (reacting with CO2) and its competitive photo-respiratory carbon oxidation (reacting with O2). RuBisCo enzyme with an enhanced CO2/O2 specificity would boost the ability to make great progress in agricultural production and environmental management. RuBisCos in marine non-green algae, resulting from an earlier endo-symbiotic event, diverge greatly from those in green plants and cyanobacteria and, further, have the highest CO2/O2 specificity whereas RuBisCos in cyanobacteria have the lowest. We assumed that there exist different levels of CO2/O2 specificity-determining factors, corresponding to different evolutionary events and specificity levels. Based on this assumption, we devised a scheme to identify these substrate-determining factors. From this analysis, we are able to discover different categories of the CO2/O2 specificity-determining factors that show which residue substitutions account for (relatively) small specificity changes, as happened in green plants, or a tremendous enhancement, as observed in marine non-green algae. Therefore, the analysis can improve our understanding of molecular mechanisms in the substrate specificity development and prioritize candidate specificity-determining surface residues for site-directed mutagenesis.