Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen Trypanosoma brucei brucei

Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis, or sleeping sickness. The causative parasite, Trypanosoma, encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. The latter are acidic membrane-enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg²⁺ and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from Tb_bVSP1 crystal structure is further confirmed by SAXS and TEM data. SAXS data suggest structural flexibility in EF hand domains indicative of conformational plasticity within Tb_bVSP1.     

Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus

Carbohydrate polymers are medically and industrially important. The S-layer of many Gram-positive organisms comprises protein and carbohydrate polymers and forms an almost paracrystalline array on the cell surface. Not only is this array important for the bacteria but it has potential application in the manufacture of commercially important polysaccharides and glycoconjugates as well. The S-layer glycoprotein glycan from Geobacillus stearothermophilus NRS 2004/3a is mainly composed of repeating units of three rhamnose sugars linked by α-1,3-, α-1,2-, and β-1,2-linkages. The formation of the β-1,2-linkage is catalysed by the enzyme WsaF. The rational use of this system is hampered by the fact that WsaF and other enzymes in the pathway share very little homology to other enzymes. We report the structural and biochemical characterisation of WsaF, the first such rhamnosyltransferase to be characterised. Structural work was aided by the surface entropy reduction method. The enzyme has two domains, the N-terminal domain, which binds the acceptor (the growing rhamnan chain), and the C-terminal domain, which binds the substrate (dTDP-β-l-rhamnose). The structure of WsaF bound to dTDP and dTDP-β-l-rhamnose coupled to biochemical analysis identifies the residues that underlie catalysis and substrate recognition. We have constructed and tested by site-directed mutagenesis a model for acceptor recognition.     

Structural Studies of Potassium Transport Protein KtrA Regulator of Conductance of K+ (RCK) C Domain in Complex with Cyclic Diadenosine Monophosphate (c-di-AMP)

Although it was only recently identified as a second messenger, c-di-AMP was found to have fundamental importance in numerous bacterial functions such as ion transport. The potassium transporter protein, KtrA, was identified as a c-di-AMP receptor. However, the co-crystallization of c-di-AMP with the protein has not been studied. Here, we determined the crystal structure of the KtrA RCK_C domain in complex with c-di-AMP. The c-di-AMP nucleotide, which adopts a U-shaped conformation, is bound at the dimer interface of RCK_C close to helices α3 and α4. c-di-AMP interacts with KtrA RCK_C mainly by forming hydrogen bonds and hydrophobic interactions. c-di-AMP binding induces the contraction of the dimer, bringing the two monomers of KtrA RCK_C into close proximity. The KtrA RCK_C was able to interact with only c-di-AMP, but not with c-di-GMP, 3′,3-cGAMP, ATP, and ADP. The structure of the KtrA RCK_C domain and c-di-AMP complex would expand our understanding about the mechanism of inactivation in Ktr transporters governed by c-di-AMP.     

Structural and Biophysical Characterization of the Interactions between Calmodulin and the Pleckstrin Homology Domain of Akt

The translocation of Akt, a serine/threonine kinase, to the plasma membrane is a critical step in the Akt activation pathway. It is established that membrane binding of Akt is mediated by direct interactions between its pleckstrin homology domain (PHD) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃). There is now evidence that Akt activation in many breast cancer cells is also modulated by the calcium-binding protein, calmodulin (CaM). Upon EGF stimulation of breast cancer cells, CaM co-localizes with Akt at the plasma membrane to enhance activation. However, the molecular details of Akt(PHD) interaction with CaM are not known. In this study, we employed NMR, biochemical, and biophysical techniques to characterize CaM binding to Akt(PHD). Our data show that CaM forms a tight complex with the PHD of Akt (dissociation constant = 100 nm). The interaction between CaM and Akt(PHD) is enthalpically driven, and the affinity is greatly dependent on salt concentration, indicating that electrostatic interactions are important for binding. The CaM-binding interface in Akt(PHD) was mapped to two loops adjacent to the PI(3,4,5)P₃ binding site, which represents a rare CaM-binding motif and suggests a synergistic relationship between CaM and PI(3,4,5)P₃ upon Akt activation. Elucidation of the mechanism by which Akt interacts with CaM will help in understanding the activation mechanism, which may provide insights for new potential targets to control the pathophysiological processes of cell survival.     

Structural Basis for the Discriminative Recognition of N6-Methyladenosine RNA by the Human YT521-B Homology Domain Family of Proteins

N⁶-Methyladenosine (m⁶A) is the most abundant internal modification in RNA and is specifically recognized by YT521-B homology (YTH) domain-containing proteins. Recently we reported that YTHDC1 prefers guanosine and disfavors adenosine at the position preceding the m⁶A nucleotide in RNA and preferentially binds to the GG(m⁶A)C sequence. Now we systematically characterized the binding affinities of the YTH domains of three other human proteins and yeast YTH domain protein Pho92 and determined the crystal structures of the YTH domains of human YTHDF1 and yeast Pho92 in complex with a 5-mer m⁶A RNA, respectively. Our binding and structural data revealed that the YTH domain used a conserved aromatic cage to recognize m⁶A. Nevertheless, none of these YTH domains, except YTHDC1, display sequence selectivity at the position preceding the m⁶A modification. Structural comparison of these different YTH domains revealed that among those, only YTHDC1 harbors a distinctly selective binding pocket for the nucleotide preceding the m⁶A nucleotide.     

Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) that converts pyruvate and coenzyme A into acetyl-CoA and formate in a reaction that is crucial to the primary metabolism of many anaerobic bacteria. The glycyl radical cofactor, which is posttranslationally installed by a radical S-adenosyl-L-methionine (SAM) activase, is a simple and effective catalyst, but is also susceptible to oxidative damage in microaerobic environments. Such damage occurs at the glycyl radical cofactor, resulting in cleaved PFL (cPFL). Bacteria have evolved a spare part protein termed YfiD that can be used to repair cPFL. Previously, we obtained a structure of YfiD by NMR spectroscopy and found that the N-terminus of YfiD was disordered and that the C-terminus of YfiD duplicates the structure of the C-terminus of PFL, including a β-strand that is not removed by the oxygen-induced cleavage. We also showed that cPFL is highly susceptible to proteolysis, suggesting that YfiD rescue of cPFL competes with protein degradation. Here, we probe the mechanism by which YfiD can bind and restore activity to cPFL through enzymatic and spectroscopic studies. Our data show that the disordered N-terminal region of YfiD is important for YfiD glycyl radical installation but not for catalysis, and that the duplicate β-strand does not need to be cleaved from cPFL for YfiD to bind. In fact, truncation of this PFL region prevents YfiD rescue. Collectively our data suggest the molecular mechanisms by which YfiD activation is precluded both when PFL is not damaged and when it is highly damaged.     

Chiral configuration of the hydration layers of D‐ and L‐alanine in water implied from dilution calorimetry

The size and configuration of the hydration layer of solutes play a major role in their thermodynamic features. With respect to amino acids in water, a series of indirect evidence strongly suggest that their hydration layer acquires a chiral configuration induced by their chiral centers. Such a chiral hydration may act as a recognition factor in the various biochemical interactions, but information on it remains rather scarce. In this study, we determined by dilution microcalorimetry the fraction of the hydration energy invested in the chiral distortion of the hydration layer surrounding D ‐ and I ‐alanine in water. The results indicate that in dilute solutions, a multilayered chiral hydration surrounds each of these solutes and amounts to over 100 water molecules. In concentrated solutions, the immediate chiral hydration layer decreases to ～30 water molecules. The energy invested in the induction of the chiral twist in the hydration layer is predominantly attributed to TΔS, the energy associated with “configurational entropy,” which amounts to only several cal/mol, about a thousandth of the total energy of the hydration shell. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Interactions of Yeast Dynein with Dynein Light Chain and Dynactin: GENERAL IMPLICATIONS FOR INTRINSICALLY DISORDERED DUPLEX SCAFFOLDS IN MULTIPROTEIN ASSEMBLIES*

Intrinsically disordered protein (IDP) duplexes composed of two IDP chains cross-linked by bivalent partner proteins form scaffolds for assembly of multiprotein complexes. The N-terminal domain of dynein intermediate chain (N-IC) is one such IDP that forms a bivalent scaffold with multiple dynein light chains including LC8, a hub protein that promotes duplex formation of diverse IDP partners. N-IC also binds a subunit of the dynein regulator, dynactin. Here we characterize interactions of a yeast ortholog of N-IC (N-Pac11) with yeast LC8 (Dyn2) or with the intermediate chain-binding subunit of yeast dynactin (Nip100). Residue level changes in Pac11 structure are monitored by NMR spectroscopy, and binding energetics are monitored by isothermal titration calorimetry (ITC). N-Pac11 is monomeric and primarily disordered except for a single α-helix (SAH) at the N terminus and a short nascent helix, LH, flanked by the two Dyn2 recognition motifs. Upon binding Dyn2, the only Pac11 residues making direct protein-protein interactions are in and immediately flanking the recognition motifs. Dyn2 binding also orders LH residues of Pac11. Upon binding Nip100, only Pac11 SAH residues make direct protein-protein interactions, but LH residues at a distant sequence position and L1 residues in an adjacent linker are also ordered. The long distance, ligand-dependent ordering of residues reveals new elements of dynamic structure within IDP linker regions.     

The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL

The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo.     

Actinosynnema mirum DSM43827溶解性多糖单加氧酶的异源表达和酶学性质表征

溶解性多糖单加氧酶(lytic polysaccharide monooxygenases,LPMO)是近年来新发现的一类作用于结晶多糖(如纤维素和几丁质)的氧化酶;LPMO通过氧化的方式来裂解底物从而有利于水解酶系进一步作用,获得可溶性寡糖。为获得更多新酶资源,通过PCR方法从Actinosynnema mirum DSM 43827菌株中成功地克隆了编码LPMO的基因Amir_5334;将该基因构建到含麦芽糖结合蛋白(maltose binding protein,MBP)标签的pET-28a(+)载体(pET-28a-MBP)上,并转化至E.coli BL21(DE3)中进行诱导表达。利用镍柱亲和层析进行纯化,获得融合表达蛋白后,使用Factor Xa蛋白酶切除MBP标签,最终得到成熟的LPMO(Am5)。成熟Am5的预测分子量约为26 kDa,等电点为8.3。分析Am5序列发现,Am5与同家族中LPMO的序列一致性较低,具有良好的序列新颖性。酶学性质分析表明Am5是对几丁质有氧化活性而对纤维素无氧化活性的LPMO;与几丁质酶协同降解几丁质时可提高几丁质酶60%的水解效率;吸附实验显示,Am5对几丁质具有较强的吸附作用,而对纤维素吸附能力较弱。以上研究表明,Am5是一种针对几丁质底物具有高效选择性的新型氧化酶。     

The effect of aminoglycoside antibiotics on the thermodynamic properties of liposomal vesicles

Liposomes are ideal drug-delivery systems because they can alter the pharmacokinetic characteristics and biodistribution profile of the incorporated bioactive molecule. The effect of the aminoglycoside antibiotics, gentamicin (GN), tobramycin (TOB), and amikacin (AMI), on the thermodynamic properties of multilamellar vesicles composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied by using differential scanning calorimetry (DSC), electron paramagnetic resonance (EPR), and ³¹P nuclear magnetic resonance (NMR) spectroscopy. The relationship between the structure of aminoglycoside antibiotics and their effect on the physical properties of the liposomal bilayers was investigated. The incorporation of the drugs was achieved and an osmotic gradient created by controlling the mole ratio of the drug inside to that outside of the DPPC vesicles so that [drug_{inside DPPC}]/[drug_{outside DPPC}] was 1:0, 1:0.2, 1:1, or 1:2.5. Incorporation of the drugs into liposomes caused the T_m to shift to a higher temperature and the δH_m and δT_1/2 values to decrease. The 2A_max and the order parameter (S), obtained from the EPR spectra, indicated that the fluidity of the liposomal membrane was affected by the type of drug and by the concentration used; GN and TOB decreased the fluidity and disturbed chain packing at mole ratios of [drug_{inside DPPC}]/[drug_{outside DPPC}] ranging from 1:0 to 1:0.2, while AMI increased the fluidity and disrupted chain packing at an osmotic gradient of 1:2.5. In conclusion, the molecular organization and thermotropic properties of the multilamellar DPPC vesicles were dependent on the osmotic gradient and structure of the aminoglycoside.     

Binding thermodynamics of paromomycin,neomycin, neomycin‐dinucleotide and ‐diPNA conjugates to bacterial and human rRNA

Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target‐drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A‐site, with two well‐known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin‐dinucleotide and ‐diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin‐diPNA conjugates as the best candidates for antimicrobial activity. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of Cys255 in HIF‐1α as a novel site for development of covalent inhibitors of HIF‐1α/ARNT PasB domain protein–protein interaction

The heterodimer HIF‐1α (hypoxia inducible factor)/HIF‐β (also known as ARNT‐aryl hydrocarbon nuclear translocator) is a key mediator of cellular response to hypoxia. The interaction between these monomer units can be modified by the action of small molecules in the binding interface between their C‐terminal heterodimerization (PasB) domains. Taking advantage of the presence of several cysteine residues located in the allosteric cavity of HIF‐1α PasB domain, we applied a cysteine‐based reactomics “hotspot identification” strategy to locate regions of HIF‐1α PasB domain critical for its interaction with ARNT. COMPOUND 5 was identified using a mass spectrometry‐based primary screening strategy and was shown to react specifically with Cys255 of the HIF‐1α PasB domain. Biophysical characterization of the interaction between PasB domains of HIF‐1α and ARNT revealed that covalent binding of COMPOUND 5 to Cys255 reduced binding affinity between HIF‐1α and ARNT PasB domains approximately 10‐fold. Detailed NMR structural analysis of HIF‐1α‐PasB‐COMPOUND 5 conjugate showed significant local conformation changes in the HIF‐1α associated with key residues involved in the HIF‐1α/ARNT PasB domain interaction as revealed by the crystal structure of the HIF‐1α/ARNT PasB heterodimer. Our screening strategy could be applied to other targets to identify pockets surrounding reactive cysteines suitable for development of small molecule modulators of protein function.     

Studies on Leaf Surface Lipid of Tobacco I. Changes in Leaf Surface Lipid and Duvatrienediol during Growth,Senescence and Curing of Tobacco Leaves

Duvatrienediol is a diterpene specifically occurred in tobacco plants and thought to be a precursor of tobacco aroma. Green tobacco leaves contained 0.2~1% of duvatrienediol per dry weight and it was corresponded to 30~60% of leaf surface lipid. Leaves on upper stalk position contained more of leaf surface lipid and duvatrienediol. In leaves on each stalk position, leaf surface lipid and duvatrienediol contents increased with leaf growth and decreased by over-maturation. Production of leaf surface lipid and duvatrienediol was affected by soil conditions or applied amount of nitrogen fertilizer. Both leaf surface lipid and duvatrienediol were decreased during curing of tobacco leaves, but the change in the latter was more drastic. Comparing to leaf surface lipid, changes in cytoplasmic lipid were less during growth and senescence of tobacco leaves.     

Comparative thermodynamic analyses of the Fv, Fab* and Fab and Fab fragments of anti-dansyl mouse monoclonal antibody

In order to investigate the role of the constant domainson the antigen-binding property of the variable domains, we have carried out a comparative thermodynamic study of the anti-dansyl Fv, Fab* and Fab fragments that possess the identical amino acid sequence of the variable domains. The thermodynamic analyses have shown that binding constants, enthalphy changes and entropy changes are similar for the three antigen-binding fragments, whereas the thermal stability of Fab is much higher than that of Fv and Fab*. We have concluded that (i) the variable domains of the three antigen-binding fragments possess identical intrinsic capability for antigen binding and (ii) the two constant domains serve to improve the stability of the variable domains.     

Dimeric c-di-GMP Is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.     

Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates

Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54_pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP.     

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinity to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 ± 0.7 × 10⁵ M⁻¹ which indicates a strong binding close to that of antibody.     

Structural and Functional Studies of the Biotin Protein Ligase from Aquifex aeolicus Reveal a Critical Role for a Conserved Residue in Target Specificity

Biotin protein ligase (BPL; EC 6.3.4.15) catalyses the formation of biotinyl-5′-AMP from biotin and ATP, and the succeeding biotinylation of the biotin carboxyl carrier protein. We describe the crystal structures, at 2.4 Å resolution, of the class I BPL from the hyperthermophilic bacteria Aquifex aeolicus (AaBPL) in its ligand-free form and in complex with biotin and ATP. The solvent-exposed β- and γ-phosphates of ATP are located in the inter-subunit cavity formed by the N- and C-terminal domains. The Arg40 residue from the conserved GXGRXG motif is shown to interact with the carboxyl group of biotin and to stabilise the α- and β-phosphates of the nucleotide. The structure of the mutant AaBPL R40G in both the ligand-free and biotin-bound forms reveals that the mutated loop has collapsed, thus hindering ATP binding. Isothermal titration calorimetry indicated that the presence of biotin is not required for ATP binding to wild-type AaBPL in the absence of Mg²⁺, and the binding of biotin and ATP has been determined to occur via a random but cooperative process. The affinity for biotin is relatively unaffected by the R40G mutation. In contrast, the thermodynamic data indicate that binding of ATP to AaBPL R40G is very weak in the absence or in the presence of biotin. The AaBPL R40G mutant remains catalytically active but shows poor substrate specificity; mass spectrometry and Western blot studies revealed that the mutant biotinylates both the target A. aeolicus BCCPΔ67 fragment and BSA, and is subject to self-biotinylation.     

Structural and Thermodynamic Characterization of the TYK2 and JAK3 Kinase Domains in Complex with CP-690550 and CMP-6

Janus kinases (JAKs) are critical regulators of cytokine pathways and attractive targets of therapeutic value in both inflammatory and myeloproliferative diseases. Although the crystal structures of active JAK1 and JAK2 kinase domains have been reported recently with the clinical compound CP-690550, the structures of both TYK2 and JAK3 with CP-690550 have remained outstanding. Here, we report the crystal structures of TYK2, a first in class structure, and JAK3 in complex with PAN-JAK inhibitors CP-690550 ((3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile) and CMP-6 (tetracyclic pyridone 2-t-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one), both of which bind in the ATP-binding cavities of both JAK isozymes in orientations similar to that observed in crystal structures of JAK1 and JAK2. Additionally, a complete thermodynamic characterization of JAK/CP-690550 complex formation was completed by isothermal titration calorimetry, indicating the critical role of the nitrile group from the CP-690550 compound. Finally, computational analysis using WaterMap further highlights the critical positioning of the CP-690550 nitrile group in the displacement of an unfavorable water molecule beneath the glycine-rich loop. Taken together, the data emphasize the outstanding properties of the kinome-selective JAK inhibitor CP-690550, as well as the challenges in obtaining JAK isozyme-selective inhibitors due to the overall structural and sequence similarities between the TYK2, JAK1, JAK2 and JAK3 isozymes. Nevertheless, subtle amino acid variations of residues lining the ligand-binding cavity of the JAK enzymes, as well as the global positioning of the glycine-rich loop, might provide the initial clues to obtaining JAK-isozyme selective inhibitors.