Promiscuous Binding Site Research Articles

State-dependent binding of cholesterol and an anionic lipid to the muscle-type Torpedo nicotinic acetylcholine receptor

The ability of the Torpedo nicotinic acetylcholine receptor (nAChR) to undergo agonist-induced conformational transitions requires the presence of cholesterol and/or anionic lipids. Here we use recently solved structures along with multiscale molecular dynamics simulations to examine lipid binding to the nAChR in bilayers that have defined effects on nAChR function. We examine how phosphatidic acid and cholesterol, lipids that support conformational transitions, individually compete for binding with phosphatidylcholine, a lipid that does not. We also examine how the two lipids work synergistically to stabilize an agonist-responsive nAChR. We identify rapidly exchanging lipid binding sites, including both phospholipid sites with a high affinity for phosphatidic acid and promiscuous cholesterol binding sites in the grooves between adjacent transmembrane α-helices. A high affinity cholesterol site is confirmed in the inner leaflet framed by a key tryptophan residue on the MX α-helix. Our data provide insight into the dynamic nature of lipid-nAChR interactions and set the stage for a detailed understanding of the mechanisms by which lipids facilitate nAChR function at the neuromuscular junction.

To bind, or not to bind, that is the question….

The unexpected findings that are described by Czub et al. [IUCrJ (2022), 9, 551-561] provide a very interesting study demonstrating how small differences in structure can result in significant changes in the relative affinities of the seemingly promiscuous binding sites that are seen with serum albumins.

The base and root of domain II loops of Cry toxins contribute to binding to Bombyx mori ABC transporter C2.

Little information is available regarding the region of Cry toxins involved in binding to their major receptors, the ATP-binding cassette (ABC) transporters. We analyzed which Cry1Aa amino acid residues contribute to binding to Bombyx mori ABC transporter C2 (BmABCC2). Several two oxidized double-cysteine substitution mutant toxins were made. In these, two amino acids at distant positions on toxin loop α8 and loop 2 or loop 2 and loop 3 were substituted with cysteine residues and crosslinked. These mutants exhibited a marked reduction in binding affinity to BmABCC2, suggesting that the binding site comprises complex cavities formed by loops α8, 2, and 3. Loop swapping between Cry1Aa and other BmABCC2-incompatible toxins indicated that loop 2 acts as a binding affinity-generating part of Cry1Aa toxin. Using single amino acid substitution mutants, the results of surface plasmon resonance (SPR) analysis and response assays with BmABCC2-expressing Sf9 cells indicated that Y366, R367, R368, and L447 in the Cry1Aa root and base region of loops 2 and 3 play important roles in binding. Furthermore, SPR analyses of these mutants suggested that a two-state binding model fits best the data obtained. Moreover, complex cavities and the above-mentioned amino acid residues contribute to the generation of multiple binding points and high-affinity binding. Finally, we found that the binding site of B. mori cadherin-like protein consists of complex cavities comprising loops 1, 2, and 3, partially overlapping that of BmABCC2, suggesting that the loop region of Cry1Aa toxin acts as a promiscuous binding site.

Microbiota Signals during the Neonatal Period Forge Life-Long Immune Responses.

The microbiota regulates immunological development during early human life, with long-term effects on health and disease. Microbial products include short-chain fatty acids (SCFAs), formyl peptides (FPs), polysaccharide A (PSA), polyamines (PAs), sphingolipids (SLPs) and aryl hydrocarbon receptor (AhR) ligands. Anti-inflammatory SCFAs are produced by Actinobacteria, Bacteroidetes, Firmicutes, Spirochaetes and Verrucomicrobia by undigested-carbohydrate fermentation. Thus, fiber amount and type determine their occurrence. FPs bind receptors from the pattern recognition family, those from commensal bacteria induce a different response than those from pathogens. PSA is a capsular polysaccharide from B. fragilis stimulating immunoregulatory protein expression, promoting IL-2, STAT1 and STAT4 gene expression, affecting cytokine production and response modulation. PAs interact with neonatal immunity, contribute to gut maturation, modulate the gut–brain axis and regulate host immunity. SLPs are composed of a sphingoid attached to a fatty acid. Prokaryotic SLPs are mostly found in anaerobes. SLPs are involved in proliferation, apoptosis and immune regulation as signaling molecules. The AhR is a transcription factor regulating development, reproduction and metabolism. AhR binds many ligands due to its promiscuous binding site. It participates in immune tolerance, involving lymphocytes and antigen-presenting cells during early development in exposed humans.

AMPA Receptor Noncompetitive Inhibitors Occupy a Promiscuous Binding Site.

Noncompetitive inhibitors of AMPA receptors have attracted interest in recent years as antiepileptic drugs. However, their development is hindered by a lack of detailed understanding of the protein-inhibitor interaction mechanisms. Recently, structures of AMPA receptor complexes with the structurally dissimilar, noncompetitive, small-molecule inhibitors pyridone perampanel (PMP), GYKI 53655 (GYKI), and CP 465022 (CP) were resolved, revealing that all three share a common binding site. However, due to the low resolution of the ligands, their exact binding modes and protein-ligand interactions remain ambiguous and insufficiently detailed. We carried out molecular dynamics (MD) simulations on X-ray-resolved and docked AMPA receptor complexes, including thermodynamic integration (TI) to compute ligand binding constants, in order to investigate the inhibitor binding modes in detail and identify key protein-ligand interaction mechanisms. Our analysis and simulations show that the ligand binding pocket at the interface of the receptor's transmembrane domain exhibits features also found in the binding pockets of the multidrug-resistance proteins. The inhibitors bind to such promiscuous pockets by forming multiple weak contacts, while the large, flexible pocket undergoes adjustments to accommodate structurally different ligands in different orientations. TI was able to identify a specific more favorable binding mode for GYKI, while PMP, which has a symmetric ring structure, produced several comparable poses indicating that it may bind in several orientations.

High Impact: The Role of Promiscuous Binding Sites in Polypharmacology.

The literature focuses on drug promiscuity, which is a drug’s ability to bind to several targets, because it plays an essential role in polypharmacology. However, little work has been completed regarding binding site promiscuity, even though its properties are now recognized among the key factors that impact drug promiscuity. Here, we quantified and characterized the promiscuity of druggable binding sites from protein-ligand complexes in the high quality Mother Of All Databases while using statistical methods. Most of the sites (80%) exhibited promiscuity, irrespective of the protein class. Nearly half were highly promiscuous and able to interact with various types of ligands. The corresponding pockets were rather large and hydrophobic, with high sulfur atom and aliphatic residue frequencies, but few side chain atoms. Consequently, their interacting ligands can be large, rigid, and weakly hydrophilic. The selective sites that interacted with one ligand type presented less favorable pocket properties for establishing ligand contacts. Thus, their ligands were highly adaptable, small, and hydrophilic. In the dataset, the promiscuity of the site rather than the drug mainly explains the multiple interactions between the drug and target, as most ligand types are dedicated to one site. This underlines the essential contribution of binding site promiscuity to drug promiscuity between different protein classes.

Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency.

Molecular docking simulations and GRID-independent molecular descriptor (GRIND) analysis to probe stereoselective interactions of CYP3A4 inhibitors

CYP3A4 has been identified as a major enzyme involved in the metabolism of drugs and xenobiotics in the human body. It is mainly expressed in the liver and has a wide capacity to oxidize structurally and functionally diverse compounds due to its large promiscuous catalytic binding site. Inhibition of CYP3A4 often leads to undesired drug–drug interactions and toxic side effects. Previous studies have demonstrated that enantiomers of the same chiral drug might inhibit CYP3A4 with different potencies and thus, one enantiomer might have different pharmacokinetics, and metabolism as compared to its counterpart. Therefore, the use of enantiopure therapeutic agents has been advocated as a promising concept to evade drug toxicity. This emphasizes the need to investigate the molecular basis of interaction and stereoselectivity of CYP3A4. Towards this goal, various in silico models have been developed that correlate the differences in the inhibitory potencies of enantiomeric pairs with difference in their interaction pattern within the binding cavity of CYP3A4. The 3D interaction based differentiating features include two hydrogen bond acceptors at a certain distance from each other, as well as from molecular boundaries of different enantiomeric pairs. Additionally, our results demonstrated the role of a hydrogen bond donor and a hydrogen bond acceptor in stereoselective inhibition of CYP3A4. For this type of study formal consent is not required

Binding of Pollutants to Biomolecules: A Simulation Study.

A number of cases around the world have been reported where animals were found dead or dying with symptoms resembling a thiamine (vitamin B) deficiency, and for some of these, a link to pollutants has been suggested. Here, we investigate whether biomolecules involved in thiamin binding and transport could be blocked by a range of different pollutants. We used in silico docking of five compound classes (25 compounds in total) to each of five targets (prion protein, ECF-type ABC transporter, thi-box riboswitch receptor, thiamin pyrophosphokinase, and YKoF protein) and subsequently performed molecular dynamics (MD) simulations to assess the stability of the complexes. The compound classes were thiamin analogues (control), pesticides, veterinary medicines, polychlorinated biphenyls, and dioxins, all of which are prevalent in the environment to some extent. A few anthropogenic compounds were found to bind the ECF-type ABC transporter, but none binds stably to prion protein. For the riboswitch, most compounds remained in their binding pockets during 50 ns of MD simulation, indicating that RNA provides a promiscuous binding site. In both YKoF and thiamin pyrophosphokinase (TPK), most compounds remain tightly bound. However, TPK biomolecules undergo pollutant-induced conformational changes. Although most compounds are found to bind to some of these targets, a larger data set is needed along with more quantitative methods like free energy perturbation calculations before firm conclusions can be drawn. This study is in part a test bed for large-scale quantitative computational screening of interactions between biological entities and pollutant molecules.

Challenges of docking in large, flexible and promiscuous binding sites

After decades of work, the correct determination of the binding mode of a small molecule into a target protein is still a challenging problem, whose difficulty depends on: (i) the sizes of the binding site and the ligand; (ii) the flexibility of both interacting partners, and (iii) the differential solvation of bound and unbound partners. We have evaluated the performance of standard rigid(receptor)/flexible(ligand) docking approaches with respect to last-generation fully flexible docking methods to obtain reasonable poses in a very challenging case: soluble Epoxide Hydrolase (sEH), a flexible protein showing different binding sites. We found that full description of the flexibility of both protein and ligand and accurate description of solvation leads to significant improvement in the ability of docking to reproduce well known binding modes, and at the same time capture the intrinsic binding promiscuity of the protein.

Tales of Dihydrofolate Binding to R67 Dihydrofolate Reductase.

Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ∼35% were constructed. Only minor effects on kcat were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-α,γ-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The Ki for this adduct was ∼9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis.

A Disease-Causing Variant in PCNA Disrupts a Promiscuous Protein Binding Site

The eukaryotic DNA polymerase sliding clamp, proliferating cell nuclear antigen or PCNA, is a ring-shaped protein complex that surrounds DNA to act as a sliding platform for increasing processivity of cellular replicases and for coordinating various cellular pathways with DNA replication. A single point mutation, Ser228Ile, in the human PCNA gene was recently identified to cause a disease whose symptoms resemble those of DNA damage and repair disorders. The mutation lies near the binding site for most PCNA-interacting proteins. However, the structural consequences of the S228I mutation are unknown. Here, we describe the structure of the disease-causing variant, which reveals a large conformational change that dramatically transforms the binding pocket for PCNA client proteins. We show that the mutation markedly alters the binding energetics for some client proteins, while another, p21CIP1, is only mildly affected. Structures of the disease variant bound to peptides derived from two PCNA partner proteins reveal that the binding pocket can adjust conformation to accommodate some ligands, indicating that the binding site is dynamic and pliable. Our work has implications for the plasticity of the binding site in PCNA and reveals how a disease mutation selectively alters interactions to a promiscuous binding site that is critical for DNA metabolism.

The tobacco lectin, prototype of the family of Nictaba-related proteins.

In the last decade, a new class of low abundant plant l ectins was identified. These proteins are expressed after exposure of the plant to different stress factors and changing environmental conditions, and therefore are referred to as "inducible" lectins. Interestingly, these lectins accumulate in the nucleocytoplasmic compartment of plant cells. At present at least six carbohydrate recognition domains have been identified within the group of nucleocytoplasmic plant lectins. This review will focus on a group of proteins that show homology to the Nicotiana tabacum (tobacco) agglutinin or Nictaba. The tobacco lectin is a 38 kDa nucleocytoplasmic protein which is only expressed upon treatment with jasmonate-related compounds or after insect herbivory. The lectin exhibits specificity towards GlcNAc, but also reacts with N-glycan structures. Extensive searches revealed that Nictaba-related sequences are widespread in the plant kingdom. Analyses of the different transcriptome databases showed that the Nictaba domain is often part of chimeric proteins comprising one or more Nictaba domain(s) fused to unrelated N- and C-terminal domains with (un)known function. At present only few proteins of these Nictaba-related proteins have been studied and characterized for their biological properties and physiological role. Despite the sequence similarity and the conserved amino acids constituting the binding site, the Nictaba domain has a promiscuous carbohydrate binding site capable of interacting with different carbohydrate motifs, suggesting that subtle changes in the vicinity of the binding site can alter its sugar specificity.

Which metal or ion? Identification of metals and ions in protein structures

Identification of ligands in single crystals of biomolecules is often uneasy. The target molecules undergo expression, purification, and special pre-crystallization treatment and even trace impurities in known chemicals become a source of potential ligands. The final "binders" belong to a large set of both natural cofactors and chemicals encountered before the diffraction experiment. Some promiscuous metal binding sites can bind different types of metals of the fourth period and activity towards the same substrate can be measured with different divalent metals in active sites of nucleases, anhydrolases, etc. Therefore, 1) Not all structures have correct ions; 2) Ion identification in all structures requires high attention, especially in metal-dependent proteins. To date 98% of X-ray structures in the PDB have the high diffraction limit of 1.2 Å or worse. Thus we must rely on other indicators as for the ion type than just the height of electron density because our data often provide an accurate picture of a mixture of states. Therefore, essential determinants (relative heights of unbiased electron density maxima, anomalous signal, the shortest interatomic distances, stability of atoms in refinement, nature of coordinating atoms) must be distinguished from marginal factors which can be smeared by worse data quality, resolution limits, position in protein chain or local disorder (atomic displacement parameters, occurrence of longer coordination distances, missing vertices of the first coordination sphere). We have applied the described approach to identify cations, such as Cu2+, Mn2+, Zn2+, Ni2+, Ca2+, Mg2+, Na+ and other ionic ligands, for instance Cl-, PO43-, SO42- [1]. The available tools for identification of metals/ions also include statistics on coordination and bonding distances [2], absorption edge detection and fluorescence [1] or microbeam proton induced X-ray emission [3]. The work was supported by MEYS CR (EE2.3.30.0029) and BIOCEV (CZ.1.05/1.1.00/02.0109).

Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles

The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein–protein interaction prediction and design methods.

From Surface to Nuclear Receptors: The Endocannabinoid Family Extends its Assets

Peroxisome proliferator-activated receptors (PPARs) have long been known as mediators of several physiological functions, among which the best characterized are lipid metabolism, energy balance and anti-inflammation. Their rather large and promiscuous ligand binding site has been recently discovered to accommodate, among a plethora of lipid molecules and metabolic intermediates, endocannabinoids and their cognate compounds, specifically belonging to the Nacylethanolamine group. In fact, oleoylethanolamide, palmitoylethanolamide and probably anandamide bind with relatively high affinity to PPARs and have now been included among their endogenous ligands. Through activation of PPARs these molecules exert a variety of physiological processes. Particularly, both long-term effects via genomic mechanisms and rapid non-genomic actions have been described, which in several instances are opposite to those evoked by activation of "classical" surface cannabinoid receptors. In this review, we describe how these effects are relevant under diverse physiological and pathophysiological circumstances, such as lipid metabolism and feeding behaviour, neuroprotection and epilepsy, circadian rhythms, addiction and cognition. A picture is emerging where nuclear receptors are involved in anorexiant, anti-inflammatory, neuroprotective, anti-epileptic, wakefulness- and cognitive-enhancing, and anti-addicting properties of endocannabinoid-like molecules. Further studies are necessary to fully understand cellular mechanisms underlying the interactions between endocannabinoids and PPARs, but also between their surface and nuclear receptors, and to exploit their potential therapeutic applications.

Energetic determinants of protein binding specificity: insights into protein interaction networks.

One of the challenges of the postgenomic era is to provide a more realistic representation of cellular processes by combining a systems biology description of functional networks with information on their interacting components. Here we carried out a systematic large-scale computational study on a structural protein-protein interaction network dataset in order to dissect thermodynamic characteristics of binding determining the interplay between protein affinity and specificity. As expected, interactions involving specific binding sites display higher affinities than those of promiscuous binding sites. Next, in order to investigate a possible role of modular distribution of hot spots in binding specificity, we divided binding sites into modules previously shown to be energetically independent. In general, hot spots that interact with different partners are located in different modules. We further observed that common hot spots tend to interact with partners exhibiting common binding motifs, whereas different hot spots tend to interact with partners with different motifs. Thus, energetic properties of binding sites provide insights into the way proteins modulate interactions with different partners. Knowledge of those factors playing a role in protein specificity is important for understanding how proteins acquire additional partners during evolution. It should also be useful in drug design.

The aryl hydrocarbon receptor: a regulator of Th17 and Treg cell development in disease

The aryl hydrocarbon receptor (AhR) was discovered almost 30 years ago as a specific binding site for the haloge-nated polycyclic aromatic hydrocarbon, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental toxin (as reviewed in [1]). Within the last decade, AhR was found to have a basic helix-loop-helix and function as a ligand-ac-tivated transcription factor. Located in the cytoplasm of most cells, AhR forms a receptor complex with several proteins including the chaperone protein hsp90 (a 90kDa heat shock protein). Once a ligand binds AhR, the ligand-receptor complex translocates to the nucleus and hsp90 is exchanged for the AhR nuclear translocator (Arnt) to form a heterodi-mer. This heterodimer binds to a DNA recognition site within a gene promoter and activates the expression of several AhR-responsive genes, including pros-taglandin synthase H2 (which converts arachidonic acid into prostaglandins) and the xenobiotic metabolizing en-zyme, CYP1A1 (cytochrome P450) .The AhR has a promiscuous ligand binding site, wherein structurally di-verse synthetic and naturally occurring AhR ligands have been identified. A review by Denison and Nagy divides AhR ligands into those formed from nonbiological activity (synthetic) and those derived from biological systems as a result of natural processes (natural) [2]. TCDD falls into the synthetic cat-egory which includes planar hydropho-bic halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. The natural category includes plant-derived materials, such as flavonoids and byproducts derived from converted dietary indoles (i.e., indole 3-carbinol and tryptophan). One such tryptophan photoproduct, 6-formylindolo[3,2-b]carbazole (FICZ), is a chemical with significant structural similarity to a high affinity natural AhR ligand, ICZ [2] .Only recently was TCCD reported to directly induce functional activation of AhR in T cells [3]. Moreover, in 2005, Nohara

Cover Picture: Catalytic Promiscuity of Halohydrin Dehalogenase and its Application in Enantioselective Epoxide Ring Opening (ChemBioChem 7/2008)

The cover picture shows a close-up of the promiscuous halide binding site of halohydrin dehalogenase, an enzyme that catalyzes enantioselective epoxide ring opening with a diversity of anionic nucleophiles. Also shown are conversions that give good yields of building blocks for a range of useful, highly enantioenriched, chiral compounds, including cyanoalcohols, nitroalcohols, and oxazolidinones. These have potential applications in agrochemicals, pharmaceuticals, and polymer chemistry. In the background, recombinant dehalogenase-producing E. coli colonies appear violet on an eosin–methylene blue indicator plate when exposed to a chloroalcohol substrate. For more information, see the communication by D. B. Janssen et al. on p. 1048 ff. of this issue.

Design of Multi-Specificity in Protein Interfaces

Interactions in protein networks may place constraints on protein interface sequences to maintain correct and avoid unwanted interactions. Here we describe a “multi-constraint” protein design protocol to predict sequences optimized for multiple criteria, such as maintaining sets of interactions, and apply it to characterize the mechanism and extent to which 20 multi-specific proteins are constrained by binding to multiple partners. We find that multi-specific binding is accommodated by at least two distinct patterns. In the simplest case, all partners share key interactions, and sequences optimized for binding to either single or multiple partners recover only a subset of native amino acid residues as optimal. More interestingly, for signaling interfaces functioning as network “hubs,” we identify a different, “multi-faceted” mode, where each binding partner prefers its own subset of wild-type residues within the promiscuous binding site. Here, integration of preferences across all partners results in sequences much more “native-like” than seen in optimization for any single binding partner alone, suggesting these interfaces are substantially optimized for multi-specificity. The two strategies make distinct predictions for interface evolution and design. Shared interfaces may be better small molecule targets, whereas multi-faceted interactions may be more “designable” for altered specificity patterns. The computational methodology presented here is generalizable for examining how naturally occurring protein sequences have been selected to satisfy a variety of positive and negative constraints, as well as for rationally designing proteins to have desired patterns of altered specificity.