The missing part: the Archaeoglobus fulgidus Argonaute forms a functional heterodimer with an N-L1-L2 domain protein.

Argonaute (Ago) proteins are present in all three domains of life (bacteria, archaea and eukaryotes). They use small (15-30 nucleotides) oligonucleotide guides to bind complementary nucleic acid targets and are responsible for gene expression regulation, mobile genome element silencing, and defence against viruses or plasmids. According to their domain organization, Agos are divided into long and short Agos. Long Agos found in prokaryotes (long-A and long-B pAgos) and eukaryotes (eAgos) comprise four major functional domains (N, PAZ, MID and PIWI) and two structural linker domains L1 and L2. The majority (∼60%) of pAgos are short pAgos, containing only the MID and inactive PIWI domains. Here we focus on the prokaryotic Argonaute AfAgo from Archaeoglobus fulgidus DSM4304. Although phylogenetically classified as a long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains, akin to short pAgos. We show that AfAgo forms a heterodimeric complex with a protein encoded upstream in the same operon, which is a structural equivalent of the N-L1-L2 domains of long pAgos. This complex, structurally equivalent to a long PAZ-less pAgo, outperforms standalone AfAgo in guide RNA-mediated target DNA binding. Our findings provide a missing piece to one of the first and the most studied pAgos.

Structural basis for sequence-specific recognition of guide and target strands by the Archaeoglobus fulgidus Argonaute protein.

Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos). Being the structural core of RNA interference machinery, they use guide RNA molecules for RNA targeting. Prokaryotic Argonautes (pAgos) are more diverse, both in terms of structure (there are eAgo-like 'long' and truncated 'short' pAgos) and mechanism, as many pAgos are specific for DNA, not RNA guide and/or target strands. Some long pAgos act as antiviral defence systems. Their defensive role was recently demonstrated for short pAgo-encoding systems SPARTA and GsSir2/Ago, but the function and action mechanisms of all other short pAgos remain unknown. In this work, we focus on the guide and target strand preferences of AfAgo, a truncated long-B Argonaute protein encoded by an archaeon Archaeoglobus fulgidus. We demonstrate that AfAgo associates with small RNA molecules carrying 5'-terminal AUU nucleotides in vivo, and characterize its affinity to various RNA and DNA guide/target strands in vitro. We also present X-ray structures of AfAgo bound to oligoduplex DNAs that provide atomic details for base-specific AfAgo interactions with both guide and target strands. Our findings broaden the range of currently known Argonaute-nucleic acid recognition mechanisms.

Short prokaryotic Argonautes provide defence against incoming mobile genetic elements through NAD+ depletion.

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). The majority (~60%) of pAgos identified bioinformatically are shorter (comprising only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here we show that Geobacter sulfurreducens short pAgo and the NAD+-bound Sir2 protein form a stable heterodimeric complex. The GsSir2/Ago complex presumably recognizes invading plasmid or phage DNA and activates the Sir2 subunit, which triggers endogenous NAD+ depletion and cell death, and prevents the propagation of invading DNA. We reconstituted NAD+ depletion activity in vitro and showed that activated GsSir2/Ago complex functions as a NADase that hydrolyses NAD+ to ADPR. Thus, short Sir2-associated pAgos provide defence against phages and plasmids, underscoring the diversity of mechanisms of prokaryotic Agos.

Beta and Gamma Amino Acid-Substituted Benzenesulfonamides as Inhibitors of Human Carbonic Anhydrases.

A series of novel benzenesulfonamide derivatives were synthesized bearing para-N ß,γ-amino acid or para-N ß-amino acid and thiazole moieties and their binding to the human carbonic anhydrase (CA) isozymes determined. These enzymes are involved in various illnesses, such as glaucoma, altitude sickness, epilepsy, obesity, and even cancer. There are numerous compounds that are inhibitors of CA and used as pharmaceuticals. However, most of them bind to most CA isozymes with little selectivity. The design of high affinity and selectivity towards one CA isozyme remains a significant challenge. The beta and gamma amino acid-substituted compound affinities were determined by the fluorescent thermal shift assay and isothermal titration calorimetry for all 12 catalytically active human carbonic anhydrase isozymes, showing the full affinity and selectivity profile. The structures of several compounds were determined by X-ray crystallography, and the binding mode in the active site of CA enzyme was shown.

Structure and mechanism of secondary sulfonamide binding to carbonic anhydrases.

Zinc-containing metalloenzyme carbonic anhydrase (CA) binds primary sulfonamides with extremely high, up to picomolar, affinity by forming a coordination bond between the negatively charged amino group and the zinc ion and making hydrogen bonds and hydrophobic contacts with other parts of the inhibitor molecule. However, N-methyl-substituted, secondary or tertiary sulfonamides bind CA with much lower affinity. In search for an explanation for this diminished affinity, a series of secondary sulfonamides were synthesized and, together with analogous primary sulfonamides, the affinities for 12 recombinant catalytically active human CA isoforms were determined by the fluorescent thermal shift assay, stopped-flow assay of the inhibition of enzymatic activity and isothermal titration calorimetry. The binding profile of secondary sulfonamides as a function of pH showed the same U-shape dependence seen for primary sulfonamides. This dependence demonstrated that there were protein binding-linked protonation reactions that should be dissected for the estimation of the intrinsic binding constants to perform structure-thermodynamics analysis. X-ray crystallographic structures of secondary sulfonamides and computational modeling dissected the atomic contributions to the binding energetics. Secondary sulfonamides bind to carbonic anhydrases via coordination bond between the negatively charged nitrogen of alkylated amino group and Zn(II) in the active site of CA. The binding reaction is linked to deprotonation of the amino group and protonation of the Zn(II)-bound hydroxide. To perform the structure-thermodynamics analysis, contributions of these linked reactions must be subtracted to determine the intrinsic energetics. In this aspect, the secondary sulfonamides are similar to primary sulfonamides as CA inhibitors.

Crystal structure of restriction endonuclease Kpn2I of CCGG-family.

BACKGROUND: Restriction endonucleases belong to prokaryotic restriction-modification systems, that protect host cells from invading DNA. Type II restriction endonucleases recognize short 4-8 bp sequences in the target DNA and cut both DNA strands producing double strand breaks. Type II restriction endonuclease Kpn2I cleaves 5'-T/CCGGA DNA sequence ("/" marks the cleavage position). Analysis of protein sequences suggested that Kpn2I belongs to the CCGG-family, which contains ten enzymes that recognize diverse nucleotides outside the conserved 5'-CCGG core and share similar motifs for the 5'-CCGG recognition and cleavage. METHODS: We solved a crystal structure of Kpn2I in a DNA-free form at 2.88 Å resolution. From the crystal structure we predicted active center and DNA recognition residues and tested them by mutational analysis. We estimated oligomeric state of Kpn2I by SEC-MALS and performed plasmid DNA cleavage assay to elucidate DNA cleavage mechanism. RESULTS: Structure comparison confirmed that Kpn2I shares a conserved active site and structural determinants for the 5'-CCGG tetranucleotide recognition with other restriction endonucleases of the CCGG-family. Guided by structural similarity between Kpn2I and the CCGG-family restriction endonucleases PfoI and AgeI, Kpn2I residues involved in the outer base pair recognition were proposed. CONCLUSIONS: Kpn2I is an orthodox Type IIP restriction endonuclease, which acts as a dimer. Kpn2I shares structural similarity to the CCGG-family restriction endonucleases PfoI, AgeI and PspGI. GENERAL SIGNIFICANCE: The Kpn2I structure concluded the studies of the CCGG-family, covering detailed structural and biochemical characterization of eleven restriction enzymes and their complexes with DNA.

Switching the Inhibitor-Enzyme Recognition Profile via Chimeric Carbonic Anhydrase XII.

A key part of the optimization of small molecules in pharmaceutical inhibitor development is to vary the molecular design to enhance complementarity of chemical features of the compound with the positioning of amino acids in the active site of a target enzyme. Typically this involves iterations of synthesis, to modify the compound, and biophysical assay, to assess the outcomes. Selective targeting of the anti-cancer carbonic anhydrase isoform XII (CA XII), this process is challenging because the overall fold is very similar across the twelve CA isoforms. To enhance drug development for CA XII we used a reverse engineering approach where mutation of the key six amino acids in the active site of human CA XII into the CA II isoform was performed to provide a protein chimera (chCA XII) which is amenable to structure-based compound optimization. Through determination of structural detail and affinity measurement of the interaction with over 60 compounds we observed that the compounds that bound CA XII more strongly than CA II, switched their preference and bound more strongly to the engineered chimera, chCA XII, based on CA II, but containing the 6 key amino acids from CA XII, behaved as CA XII in its compound recognition profile. The structures of the compounds in the chimeric active site also resembled those determined for complexes with CA XII, hence validating this protein engineering approach in the development of new inhibitors.

Oriented Soft DNA Curtains for Single-Molecule Imaging.

Over the past 20 years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes a flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flow-stretch assay of stably immobilized and oriented DNA molecules using a protein template-directed assembly. In our assay, a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended by either single- or both-end immobilization to the protein templates. For oriented both-end DNA immobilization, we employed heterologous DNA labeling and protein template coverage with the antidigoxigenin antibody. In contrast to single-end immobilization, both-end immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein-DNA interactions at more controllable reaction conditions. Additionally, we increased the immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g., CRISPR-Cas9) binding assay that monitors the binding location and position of individual fluorescently labeled proteins on DNA.

Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer.

Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are composed of four major structural/functional domains (N, PAZ, MID, and PIWI) and thereby closely resemble eAgos. It was demonstrated that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown. In this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal ß-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including SEC-MALS, SAXS, single-molecule FRET, and AFM, to show that AfAgo is indeed a homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding underscores the diversity of prokaryotic Agos and broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.

Methyl 2-Halo-4-Substituted-5-Sulfamoyl-Benzoates as High Affinity and Selective Inhibitors of Carbonic Anhydrase IX.

Among the twelve catalytically active carbonic anhydrase isozymes present in the human body, the CAIX is highly overexpressed in various solid tumors. The enzyme acidifies the tumor microenvironment enabling invasion and metastatic processes. Therefore, many attempts have been made to design chemical compounds that would exhibit high affinity and selective binding to CAIX over the remaining eleven catalytically active CA isozymes to limit undesired side effects. It has been postulated that such drugs may have anticancer properties and could be used in tumor treatment. Here we have designed a series of compounds, methyl 5-sulfamoyl-benzoates, which bear a primary sulfonamide group, a well-known marker of CA inhibitors, and determined their affinities for all twelve CA isozymes. Variations of substituents on the benzenesulfonamide ring led to compound 4b, which exhibited an extremely high observed binding affinity to CAIX; the Kd was 0.12 nM. The intrinsic dissociation constant, where the binding-linked protonation reactions have been subtracted, reached 0.08 pM. The compound also exhibited more than 100-fold selectivity over the remaining CA isozymes. The X-ray crystallographic structure of compound 3b bound to CAIX showed the structural position, while several structures of compounds bound to other CA isozymes showed structural reasons for compound selectivity towards CAIX. Since this series of compounds possess physicochemical properties suitable for drugs, they may be developed for anticancer therapeutic purposes.

Halogenated and di-substituted benzenesulfonamides as selective inhibitors of carbonic anhydrase isoforms.

By applying an approach of a "ring with two tails", a series of novel inhibitors possessing high-affinity and significant selectivity towards selected carbonic anhydrase (CA) isoforms has been designed. The "ring" consists of 2-chloro/bromo-benzenesulfonamide, where the sulfonamide group is as an anchor coordinating the Zn(II) in the active site of CAs, and halogen atom orients the ring affecting the affinity and selectivity. First "tail" is a substituent containing carbonyl, carboxyl, hydroxyl, ether groups or hydrophilic amide linkage. The second "tail" contains aryl- or alkyl-substituents attached through a sulfanyl or sulfonyl group. Both "tails" are connected to the benzene ring and play a crucial role in selectivity. Varying the substituents, we designed compounds selective for CA VII, CA IX, CA XII, or CA XIV. Since due to binding-linked protonation reactions the binding-ready fractions of the compound and protein are much lower than one, the "intrinsic" affinities were calculated that should be used to study correlations between crystal structures and the thermodynamics of binding for rational drug design. The "intrinsic" affinities together with the intrinsic enthalpies and entropies of binding together with co-crystal structures were used demonstrate structural factors determining major contributions for compound affinity and selectivity.

Decision-Making in Cascade Complexes Harboring crRNAs of Altered Length.

The multi-subunit type I CRISPR-Cas surveillance complex Cascade uses its crRNA to recognize dsDNA targets. Recognition involves DNA unwinding and base-pairing between the crRNA spacer region and a complementary DNA strand, resulting in formation of an R-loop structure. The modular Cascade architecture allows assembly of complexes containing crRNAs with altered spacer lengths that promise increased target specificity in emerging biotechnological applications. Here we produce type I-E Cascade complexes containing crRNAs with up to 57-nt-long spacers. We show that these complexes form R-loops corresponding to the designed target length, even for the longest spacers tested. Furthermore, the complexes can bind their targets with much higher affinity compared with the wild-type form. However, target recognition and the subsequent Cas3-mediated DNA cleavage do not require extended R-loops but already occur for wild-type-sized R-loops. These findings set important limits for specificity improvements of type I CRISPR-Cas systems.

Engineered Carbonic Anhydrase VI-Mimic Enzyme Switched the Structure and Affinities of Inhibitors.

Secretory human carbonic anhydrase VI (CA VI) has emerged as a potential drug target due to its role in pathological states, such as excess acidity-caused dental caries and injuries of gastric epithelium. Currently, there are no available CA VI-selective inhibitors or crystallographic structures of inhibitors bound to CA VI. The present study focuses on the site-directed CA II mutant mimicking the active site of CA VI for inhibitor screening. The interactions between CA VI-mimic and a series of benzenesulfonamides were evaluated by fluorescent thermal shift assay, stopped-flow CO2 hydration assay, isothermal titration calorimetry, and X-ray crystallography. Kinetic parameters showed that A65T, N67Q, F130Y, V134Q, L203T mutations did not influence catalytic properties of CA II, but inhibitor affinities resembled CA VI, exhibiting up to 0.16 nM intrinsic affinity for CA VI-mimic. Structurally, binding site of CA VI-mimic was found to be similar to CA VI. The ligand interactions with mutated side chains observed in three crystallographic structures allowed to rationalize observed variation of binding modes and experimental binding affinities to CA VI. This integrative set of kinetic, thermodynamic, and structural data revealed CA VI-mimic as a useful model to design CA VI-specific inhibitors which could be beneficial for novel therapeutic applications.

Fixed DNA Molecule Arrays for High-Throughput Single DNA-Protein Interaction Studies.

The DNA Curtains assay is a recently developed experimental platform for protein-DNA interaction studies at the single-molecule level that is based on anchoring and alignment of DNA fragments. The DNA Curtains so far have been made by using chromium barriers and fluid lipid bilayer membranes, which makes such a specialized assay technically challenging and relatively unstable. Herein, we report on an alternative strategy for DNA arraying for analysis of individual DNA-protein interactions. It relies on stable DNA tethering onto nanopatterned protein templates via high affinity molecular recognition. We describe fabrication of streptavidin templates (line features as narrow as 200 nm) onto modified glass coverslips by combining surface chemistry, atomic force microscopy (AFM), and soft lithography techniques with affinity-driven assembly. We have employed such chips for arraying single- and double-tethered DNA strands, and we characterized the obtained molecular architecture: we evaluated the structural characteristics and specific versus nonspecific binding of fluorescence-labeled DNA using AFM and total internal reflection fluorescence microscopy. We demonstrate the feasibility of our DNA molecule arrays for short single-tethered as well as for lambda single- and double-tethered DNA. The latter type of arrays proved very suitable for localization of single DNA-protein interactions employing restriction endonucleases. The presented molecular architecture and facile method of fabrication of our nanoscale platform does not require clean room equipment, and it offers advanced functional studies of DNA machineries and the development of future nanodevices.

Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family.

Restriction endonucleases (REs) of the CCGG-family recognize a set of 4-8 bp target sequences that share a common CCGG or CCNGG core and possess PD…D/ExK nuclease fold. REs that interact with 5 bp sequence 5'-CCNGG flip the central N nucleotides and 'compress' the bound DNA to stack the inner base pairs to mimic the CCGG sequence. PfoI belongs to the CCGG-family and cleaves the 7 bp sequence 5'-T|CCNGGA ("|" designates cleavage position). We present here crystal structures of PfoI in free and DNA-bound forms that show unique active site arrangement and mechanism of sequence recognition. Structures and mutagenesis indicate that PfoI features a permuted E…ExD…K active site that differs from the consensus motif characteristic to other family members. Although PfoI also flips the central N nucleotides of the target sequence it does not 'compress' the bound DNA. Instead, PfoI induces a drastic change in DNA backbone conformation that shortens the distance between scissile phosphates to match that in the unperturbed CCGG sequence. Our data demonstrate the diversity and versatility of structural mechanisms employed by restriction enzymes for recognition of related DNA sequences.

DNA recognition by Arabidopsis transcription factors ABI3 and NGA1.

B3 transcription factors constitute a large plant-specific protein superfamily, which plays a central role in plant life. Family members are characterized by the presence of B3 DNA-binding domains (DBDs). To date, only a few B3 DBDs were structurally characterized; therefore, the DNA recognition mechanism of other family members remains to be elucidated. Here, we analyze DNA recognition mechanism of two structurally uncharacterized B3 transcription factors, ABI3 and NGA1. Guided by the structure of the DNA-bound B3 domain of Arabidopsis transcriptional repressor VAL1, we have performed mutational analysis of the ABI3 B3 domain. We demonstrate that both VAL1-B3 and ABI3-B3 recognize the Sph/RY DNA sequence 5'-TGCATG-3' via a conserved set of base-specific contacts. We have also solved a 1.8 Å apo-structure of NGA1-B3, DBD of Arabidopsis transcription factor NGA1. We show that NGA1-B3, like the structurally related RAV1-B3 domain, is specific for the 5'-CACCTG-3' DNA sequence, albeit tolerates single base pair substitutions at the 5'-terminal half of the recognition site. Employing distance-dependent fluorophore quenching, we show that NGA1-B3 binds the asymmetric recognition site in a defined orientation, with the 'N-arm' and 'C-arm' structural elements interacting with the 5'- and 3'-terminal nucleotides of the 5'-CACCTG-3' sequence, respectively. Mutational analysis guided by the model of DNA-bound NGA1-B3 helped us identify NGA1-B3 residues involved in base-specific and DNA backbone contacts, providing new insights into the mechanism of DNA recognition by plant B3 domains of RAV and REM families. DATABASES: RCSB Protein Data Bank, accession number 5OS9.

Recognition of modified cytosine variants by the DNA-binding domain of methyl-directed endonuclease McrBC.

Cytosine modifications expand the information content of genomic DNA in both eukaryotes and prokaryotes, providing means for epigenetic regulation and self versus nonself discrimination. For example, the methyl-directed restriction endonuclease, McrBC, recognizes and cuts invading bacteriophage DNA containing 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and N4-methylcytosine (4mC), leaving the unmodified host DNA intact. Here, we present cocrystal structures of McrB-N bound to DNA oligoduplexes containing 5hmC, 5-formylcytosine (5fC), and 4mC, and characterize the relative affinity of McrB-N to various cytosine variants. We find that McrB-N flips out modified bases into a protein pocket and binds cytosine derivatives in the order of descending affinity: 4mC > 5mC > 5hmC â‰« 5fC. We also show that pocket mutations alter the relative preference of McrB-N to 5mC, 5hmC, and 4mC.

Activity and structure of EcoKMcrA.

Escherichia coli McrA (EcoKMcrA) acts as a methylcytosine and hydroxymethylcytosine dependent restriction endonuclease. We present a biochemical characterization of EcoKMcrA that includes the first demonstration of its endonuclease activity, small angle X-ray scattering (SAXS) data, and a crystal structure of the enzyme in the absence of DNA. Our data indicate that EcoKMcrA dimerizes via the anticipated C-terminal HNH domains, which together form a single DNA binding site. The N-terminal domains are not homologous to SRA domains, do not interact with each other, and have separate DNA binding sites. Electrophoretic mobility shift assay (EMSA) and footprinting experiments suggest that the N-terminal domains can sense the presence and sequence context of modified cytosines. Pyrrolocytosine fluorescence data indicate no base flipping. In vitro, EcoKMcrA DNA endonuclease activity requires Mn2+ ions, is not strictly methyl dependent, and is not observed when active site variants of the enzyme are used. In cells, EcoKMcrA specifically restricts DNA that is modified in the correct sequence context. This activity is impaired by mutations of the nuclease active site, unless the enzyme is highly overexpressed.

Design of two-tail compounds with rotationally fixed benzenesulfonamide ring as inhibitors of carbonic anhydrases.

Rational design of compounds that would bind specific pockets of the target proteins is a difficult task in drug design. The 12 isoforms of catalytically active human carbonic anhydrases (CAs) have highly similar active sites that make it difficult to design inhibitors selective for one or several CA isoforms. A series of CA inhibitors based on 2-chloro/bromo-benzenesulfonamide that is largely fixed in the CA active site together with one or two tails yielded compounds that were synthesized and evaluated as inhibitors of CA isoforms. Introduction of a second tail had significant influence on the binding affinity and two-tailed compounds in most cases provided high affinity and selectivity for CA IX and CA XIV. The contacts between several compounds and CA amino acids were determined by X-ray crystallography. Together with the intrinsic enthalpy and entropy of binding they provided the structure-thermodynamics correlations for this series of compounds with the insight how to rationally build compounds with desired CA isoform as a target.

Crystal structure correlations with the intrinsic thermodynamics of human carbonic anhydrase inhibitor binding.

The structure-thermodynamics correlation analysis was performed for a series of fluorine- and chlorine-substituted benzenesulfonamide inhibitors binding to several human carbonic anhydrase (CA) isoforms. The total of 24 crystal structures of 16 inhibitors bound to isoforms CA I, CA II, CA XII, and CA XIII provided the structural information of selective recognition between a compound and CA isoform. The binding thermodynamics of all structures was determined by the analysis of binding-linked protonation events, yielding the intrinsic parameters, i.e., the enthalpy, entropy, and Gibbs energy of binding. Inhibitor binding was compared within structurally similar pairs that differ by para- or meta-substituents enabling to obtain the contributing energies of ligand fragments. The pairs were divided into two groups. First, similar binders-the pairs that keep the same orientation of the benzene ring exhibited classical hydrophobic effect, a less exothermic enthalpy and a more favorable entropy upon addition of the hydrophobic fragments. Second, dissimilar binders-the pairs of binders that demonstrated altered positions of the benzene rings exhibited the non-classical hydrophobic effect, a more favorable enthalpy and variable entropy contribution. A deeper understanding of the energies contributing to the protein-ligand recognition should lead toward the eventual goal of rational drug design where chemical structures of ligands could be designed based on the target protein structure.