A novel type of receptor protein, based on the lipocalin scaffold, with specificity for digoxigenin

We demonstrate that the bilin-binding protein, a member of the lipocalin family of proteins, can be structurally reshaped in order to specifically complex digoxigenin, a steroid ligand commonly used for the non-radioactive labelling of biomolecules. 16 amino acid residues, distributed across the four loops which form the binding site of the bilin-binding protein, were subjected to targeted random mutagenesis. From the resulting library the variant DigA16 was obtained by combined use of phage display and a filter-sandwich colony screening assay, followed by in vitro affinity maturation. DigA16 possesses strong binding activity and high specificity for the digoxigenin group, with a K(D) of 30.2(+/-3.6) nM. The derivative compound digitoxigenin is bound even more tightly, with a K(D) of 2.0(+/-0.52) nM, whereas the steroid glycoside ouabain is not recognized at all. Fusion proteins between DigA16 and alkaline phosphatase were constructed and shown to retain both the digoxigenin-binding function and enzymatic activity, irrespective of whether the enzyme was fused to the N or the C terminus of the bilin-binding protein variant. Our findings suggest that the lipocalin scaffold can be generally employed for the construction of specific receptor proteins, so-called "anticalins", which provide a promising alternative to recombinant antibody fragments.     

Tuning ligand affinity,specificity, and folding stability of an engineered lipocalin variant -- a so-called 'anticalin' -- using a molecular random approach

Anticalins are prepared by reshaping the ligand pocket of a natural lipocalin via protein engineering in order to recognize a prescribed ligand. In this manner, the anticalin DigA with specificity for digoxigenin was previously derived from the bilin-binding protein (BBP), a natural lipocalin from Pieris brassicae. The four peptide loops that form its ligand-binding site were randomized and a cognate variant was selected from the resulting library. Here, we propose a concept for improving the ligand-binding properties of this anticalin in an in vitro affinity maturation process by step-wise randomization of restricted areas of the loop region. Following selection on digoxigenin-binding activity via phage display and colony screening, several DigA variants were thus obtained. The recombinant proteins were thoroughly characterized in terms of ligand affinity and specificity, secondary structure and thermal stability against unfolding. The variant DigA16/19, which carries several new mutations, exhibits clearly improved affinity for digoxigenin, with K(D)=12.4 nM. Hence, it is suitable as a sensitive reagent in biochemical detection experiments, especially when produced as a functional fusion protein with alkaline phosphatase as reporter enzyme. In addition, DigA16/19 possesses enhanced ligand specificity and recognizes part of the linker that was used for fixing the steroid group to a carrier protein. Finally, the digoxigenin-binding anticalins appear to have high physico-chemical stability, with T(m) values in the 70 degrees C range. Our present findings support the notion that anticalins provide a useful class of compact and robust ligand-receptor proteins that can be tailored for practical demands.     

Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin

The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion. The combination of site-directed tryptophan fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X-ray crystallography.     

Steered molecular dynamics simulations of ligand–receptor interaction in lipocalins

Retinol binding protein (RBP) and an engineered lipocalin, DigA16, have been studied using molecular dynamics simulations. Special emphasis has been placed on explaining the ligand–receptor interaction in RBP–retinol and DigA16–digoxigenin complexes, and steered molecular dynamics simulations of 10–20 ns have been carried out for the ligand expulsion process. Digoxigenin is bound deep inside the cavity of DigA16 and forms several stable hydrogen bonds in addition to the hydrophobic van der Waals interaction with the aromatic side-chains. Four crystalline water molecules inside the ligand-binding cavity remain trapped during the simulations. The strongly hydrophobic receptor site of RBP differs considerably from DigA16, and the main source of ligand attraction comes from the phenyl side-chains. The hydrogen bonds between digoxigenin and DigA16 cause the rupture forces on ligand removal in DigA16 and RBP to differ. The mutated DigA16 residues contribute approximately one-half of the digoxigenin interaction energy with DigA16 and, of these, the energetically most important are residues His35, Arg58, Ser87, Tyr88, and Phe114. Potential “sensor loops” were found for both receptors. These are the outlier loops between residues 114–121 and 63–67 for DigA16 and RBP, respectively, and they are located near the entrance of the ligand-binding cavity. Especially, the residues Glu119 (DigA16) and Leu64 (RBP) are critical for sensing. The ligand binding energies have been estimated based on the linear response approximation of binding affinity by using a previous parametrization for retinoids and RBP.     

Crystallographic analysis of an "anticalin" with tailored specificity for fluorescein reveals high structural plasticity of the lipocalin loop region

The artificial lipocalin FluA with novel specificity toward fluorescein was derived via combinatorial engineering from the bilin-binding protein, BBP by exchange of 16 amino acids in the ligand pocket. Here, we describe the crystal structure of FluA at 2.0 A resolution in the space group P2(1) with two protein-ligand complexes in the asymmetric unit. In both molecules, the characteristic beta-barrel architecture with the attached alpha-helix is well preserved. In contrast, the four loops at one end of the beta-barrel that form the entrance to the binding site exhibit large conformational deviations from the wild-type protein, which can be attributed to the sidechain replacements. Specificity for the new ligand is furnished by hydrophobic packing, charged sidechain environment, and hydrogen bonds with its hydroxyl groups. Unexpectedly, fluorescein is bound in a much deeper cavity than biliverdin IX(gamma) in the natural lipocalin. Triggered by the substituted residues, unmutated sidechains at the bottom of the binding site adopt conformations that are quite different from those observed in the BBP, illustrating that not only the loop region but also the hydrophobic interior of the beta-barrel can be reshaped for molecular recognition. Particularly, Trp 129 participates in a tight stacking interaction with the xanthenolone moiety, which may explain the ultrafast electron transfer that occurs on light excitation of the bound fluorescein. These structural findings support our concept of using lipocalins as a scaffold for the engineering of so-called "anticalins" directed against prescribed targets as an alternative to recombinant antibody fragments.     

Crystal structure of a recombinant anti-estradiol Fab fragment in complex with 17beta -estradiol.

The crystal structure of a Fab fragment of an anti-17beta-estradiol antibody 57-2 was determined in the absence and presence of the steroid ligand, 17beta-estradiol (E2), at 2.5 and 2.15-A resolutions, respectively. The antibody binds the steroid in a deep hydrophobic pocket formed at the interface between the variable domains. No major structural rearrangements take place upon ligand binding; however, a large part of the heavy chain variable domain near the binding pocket is unusually flexible and is partly stabilized when the steroid is bound. The nonpolar steroid skeleton of E2 is recognized by a number of hydrophobic interactions, whereas the two hydroxyl groups of E2 are hydrogen-bonded to the protein. Especially, the 17-hydroxyl group of E2 is recognized by an intricate hydrogen bonding network in which the 17-hydroxyl itself forms a rare four-center hydrogen bond with three polar amino acids; this hydrogen bonding arrangement accounts for the low cross-reactivity of the antibody with other estrogens such as estrone. The CDRH3 loop plays a prominent role in ligand binding. All the complementarity-determining regions of the light chain make direct contacts with the steroid, even CDRL2, which is rarely directly involved in the binding of haptens.     

Side-chain flexibility in proteins upon ligand binding

Ligand binding may involve a wide range of structural changes in the receptor protein, from hinge movement of entire domains to small side-chain rearrangements in the binding pocket residues. The analysis of side chain flexibility gives insights valuable to improve docking algorithms and can provide an index of amino-acid side-chain flexibility potentially useful in molecular biology and protein engineering studies. In this study we analyzed side-chain rearrangements upon ligand binding. We constructed two non-redundant databases (980 and 353 entries) of "paired" protein structures in complexed (holo-protein) and uncomplexed (apo-protein) forms from the PDB macromolecular structural database. The number and identity of binding pocket residues that undergo side-chain conformational changes were determined. We show that, in general, only a small number of residues in the pocket undergo such changes (e.g., approximately 85% of cases show changes in three residues or less). The flexibility scale has the following order: Lys > Arg, Gln, Met > Glu, Ile, Leu > Asn, Thr, Val, Tyr, Ser, His, Asp > Cys, Trp, Phe; thus, Lys side chains in binding pockets flex 25 times more often then do the Phe side chains. Normalizing for the number of flexible dihedral bonds in each amino acid attenuates the scale somewhat, however, the clear trend of large, polar amino acids being more flexible in the pocket than aromatic ones remains. We found no correlation between backbone movement of a residue upon ligand binding and the flexibility of its side chain. These results are relevant to 1. Reduction of search space in docking algorithms by inclusion of side-chain flexibility for a limited number of binding pocket residues; and 2. Utilization of the amino acid flexibility scale in protein engineering studies to alter the flexibility of binding pockets.     

A succession of substrate induced conformational changes ensures the amino acid specificity of Thermus thermophilus prolyl-tRNA synthetase: comparison with histidyl-tRNA synthetase

We describe the recognition by Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) of proline, ATP and prolyl-adenylate and the sequential conformational changes occurring when the substrates bind and the activated intermediate is formed. Proline and ATP binding cause respectively conformational changes in the proline binding loop and motif 2 loop. However formation of the activated intermediate is necessary for the final conformational ordering of a ten residue peptide ("ordering loop") close to the active site which would appear to be essential for functional tRNA 3' end binding. These induced fit conformational changes ensure that the enzyme is highly specific for proline activation and aminoacylation. We also present new structures of apo and AMP bound histidyl-tRNA synthetase (HisRS) from T. thermophilus which we compare to our previous structures of the histidine and histidyl-adenylate bound enzyme. Qualitatively, similar results to those observed with T. thermophilus prolyl-tRNA synthetase are found. However histidine binding is sufficient to induce the co-operative ordering of the topologically equivalent histidine binding loop and ordering loop. These two examples contrast with most other class II aminoacyl-tRNA synthetases whose pocket for the cognate amino acid side-chain is largely preformed. T. thermophilus prolyl-tRNA synthetase appears to be the second class II aminoacyl-tRNA synthetase, after HisRS, to use a positively charged amino acid instead of a divalent cation to catalyse the amino acid activation reaction.     

Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors.

BACKGROUND: The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. RESULTS: We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. CONCLUSIONS: The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.     

Crystal structure of cockroach allergen Bla g 2, an unusual zinc binding aspartic protease with a novel mode of self-inhibition

The crystal structure of Bla g 2 was solved in order to investigate the structural basis for the allergenic properties of this unusual protein. This is the first structure of an aspartic protease in which conserved glycine residues, in two canonical DTG triads, are substituted by different amino acid residues. Another unprecedented feature revealed by the structure is the single phenylalanine residue insertion on the tip of the flap, with the side-chain occupying the S1 binding pocket. This and other important amino acid substitutions in the active site region of Bla g 2 modify the interactions in the vicinity of the catalytic aspartate residues, increasing the distance between them to approximately 4A and establishing unique direct contacts between the flap and the catalytic residues. We attribute the absence of substantial catalytic activity in Bla g 2 to these unusual features of the active site. Five disulfide bridges and a Zn-binding site confer stability to the protein, which may contribute to sensitization at lower levels of exposure than other allergens.     

Molecular details of Itk activation by prolyl isomerization and phospholigand binding: the NMR structure of the Itk SH2 domain bound to a phosphopeptide

The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) is a critical component of the regulatory apparatus controlling the activity of this immunologically important enzyme. To gain insight into the structural features associated with the activated form of Itk, we have solved the NMR structure of the SH2 domain bound to a phosphotyrosine-containing peptide (pY) and analyzed changes in trans-hydrogen bond scalar couplings ((3h)J(NC')) that result from pY binding. Isomerization of a single prolyl imide bond in this domain is responsible for simultaneous existence of two distinct SH2 conformers. Prolyl isomerization directs ligand recognition: the trans conformer preferentially binds pY. The structure of the SH2/pY complex provides insight into the ligand specificity; the BG loop in the ligand-free trans SH2 conformer is pre-arranged for optimal contacts with the pY+3 residue of the ligand. Analysis of (3h)J(NC') couplings arising from hydrogen bonds has revealed propagation of structural changes from the pY binding pocket to the CD loop containing conformationally heterogeneous proline as well as to the alphaB helix, on the opposite site of the domain. These findings offer a structural framework for understanding the roles of prolyl isomerization and pY binding in Itk regulation.     

Crystal structure of the human odorant binding protein,OBPIIa

Human odorant‐binding protein, OBP_IIa, is expressed by nasal epithelia to facilitate transport of hydrophobic odorant molecules across the aqueous mucus. Here, we report its crystallographic analysis at 2.6 Å resolution. OBP_IIa is a monomeric protein that exhibits the classical lipocalin fold with a conserved eight‐stranded β‐barrel harboring a remarkably large hydrophobic pocket. Basic residues within the four loops that shape the entrance to this ligand‐binding site evoke a positive electrostatic potential. Human OBP_IIa shows distinct features compared with other mammalian OBPs, including a potentially reactive Cys side chain within its pocket similar to human tear lipocalin. Proteins 2015; 83:1180–1184. © 2015 Wiley Periodicals, Inc.     

Modeling, mutagenesis, and structural studies on the fully conserved phosphate-binding loop (loop 8) of triosephosphate isomerase: toward a new substrate specificity

Loop 8 (residues 232-242) in triosephosphate isomerase (TIM) is a highly conserved loop that forms a tight binding pocket for the phosphate moiety of the substrate. Its sequence includes the fully conserved, solvent-exposed Leu238. The tight phosphate-binding pocket explains the high substrate specificity of TIM being limited to the in vivo substrates dihydroxyacetone-phosphate and D-glyceraldehyde-3-phosphate. Here we use the monomeric variant of trypanosomal TIM for exploring the structural consequences of shortening this loop. The mutagenesis, guided by extensive modeling calculations and followed up by crystallographic characterization, is aimed at widening the phosphate-binding pocket and, consequently, changing the substrate specificity. Two new variants were characterized. The crystal structures of these variants indicate that in monomeric forms of TIM, the Leu238 side-chain is nicely buried in a hydrophobic cluster. Monomeric forms of wild-type dimeric TIM are known to exist transiently as folding intermediates; our structural analysis suggests that in this monomeric form, Leu238 of loop 8 also adopts this completely buried conformation, which explains its full conservation across the evolution. The much wider phosphate-binding pocket of the new variant allows for the development of a new TIM variant with a different substrate specificity.     

Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions

The structural origins of the specificity of the neurophysin hormone-binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide alpha-amino group, two factors are considered central. (1) The bound Leu side chain can establish only one-third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition.     

A mining minima approach to exploring the docking pathways of p-nitrocatechol sulfate to YopH

Using the docking of p-nitrocatechol sulfate to Yersinia protein tyrosine phosphatase YopH as an example, we showed that an approach based on mining minima followed by cluster and similarity analysis could generate useful insights into docking pathways. Our simulation treated both the ligand and the protein as flexible molecules so that the coupling between their motion could be properly accounted for. Our simulation identified three docking poses; the one with the lowest energy agreed well with experimental structure. The model also predicted the side-chain conformations of the amino acids lying in the binding pocket correctly with the exception of three residues that appeared to be stabilized by two structural water molecules in the crystal structure. The implicit solvent model employed in the simulation could not capture such effects well. We also found four major pathways leading to these docking poses after the ligand entered the mouth of the binding pocket. In addition, the sulfate group of p-nitrocatechol sulfate was found to be important both in binding the ligand to the pocket and in guiding the ligand to dock into the pocket. The coupling of the motion between the protein and the ligand also played an important role in facilitating ligand loading and unloading.     

Screening for engineered neomycin riboswitches that control translation initiation

Riboswitches are genetic control elements that regulate gene expression in a small molecule-dependent way. We developed a two-stage strategy of in vitro selection followed by a genetic screen and identified several artificial small molecule-binding riboswitches that respond to the aminoglycoside neomycin. Structure-function relationships and structural probing revealed that they adopt the general neomycin-binding motif. They display no sequence similarities to in vitro selected neomycin aptamers but contain parts of the decoding site that is the binding site for neomycin on the ribosomal RNA. We propose a model of a composed binding pocket of an internal loop as primary docking site and a terminal flaplike loop structure fixing neomycin in a sandwich-like manner. Such binding pockets characterized by multiple contacts between ligand and RNA are described for both natural and engineered riboswitches. We anticipate that combination of in vitro selection and in vivo screening is a useful strategy to identify RNA molecules with a desired functionality.     

Improving the antigen affinity of an antibody Fv-fragment by protein design.

The affinity of an antibody for its ligand 2-phenyloxazolone was improved by protein design. For the design two-dimensional nuclear magnetic resonance spectroscopy, protein engineering and molecular modelling were used in an interactive scheme. Initially the binding site was localized with the help of transferred nuclear Overhauser enhancement signals from two, site specifically assigned tyrosine side-chains in the complementarity-determining regions of the antibody to the ligand 4-glycyl-2-phenyloxazolone. On their basis the hapten was placed into a model of the Fv-fragment built according to the principles of canonical antibody structures. From the model, unfavourable contacts between hapten and an aspartyl side-chain in complementarity-determining region 3 of the heavy chain were predicted. Substitution of the aspartyl residue by alanine resulted in a threefold increase in affinity of the antibody Fv-fragment for two hapten derivatives when compared with the wild-type. Nuclear magnetic resonance analysis of the improved Fv-fragment revealed an interaction between the alpha-carbon proton of alanyl residue with the ligand, which was not seen for the aspartyl residue. This interaction is not entirely in accordance with the model, which predicts an interaction between the side-chain of this residue and the hapten. However, it shows that by combined use of nuclear magnetic resonance analysis and molecular modelling, a residue that is critical for antigen binding was identified, whose mutation allowed the design of an improved antibody combining site.     

Interstrand loops CD and EF act as pH-dependent gates to regulate fatty acid ligand binding in tear lipocalin

Tear lipocalin (TL), a major component of human tears, shows pH-dependent endogenous ligand binding. The structural and conformational changes associated with ligand release in the pH range of 7.5-3.0 are monitored by circular dichroism spectroscopy and site-directed tryptophan fluorescence. In the transition from pH 7.5 to pH 5.5, the ligand affinity for 16-(9-anthroyloxy)palmitic acid (16AP) and 8-anilino-1-naphthalenesulfonic acid is reduced. At pH 4.0 these ligands no longer bind within the TL calyx. From pH 7.3 to pH 3.0, the residues on loops CD and EF, which overhang the calyx entrance, show reduced accessibility to acrylamide. In addition resonance energy transfer is enhanced between residues on the two loops; the distance between the loops narrows. These findings suggest that apposition of the loops at low pH excludes the ligand from the intracavitary binding site. The conformational changes observed in transition from pH 7.3 to pH 3.0 for loops CD and EF are quite different. The CD loop shows less population reshuffling than the EF loop with an acidic environment, probably because backbone motion is restrained by the adjacent disulfide bond. The Trp fluorescence wavelength maximum (lambda(max)) reflects internal electrostatic interactions for positions on loops CD and EF. The titration curves of lambda(max) for mutants on the EF loop fit the Hendersen-Hasselbalch equation for two apparent pK(a) values, while the CD loop positions fit satisfactorily with one pK(a) value. Midpoints of transition for the binding affinity of TL tryptophan mutants to 16AP occur at pH 5.5-6.1. Replacement of each amino acid on either loop by single tryptophan mutation does not disrupt the pH-dependent binding affinity to 16AP. Taken together the data suggest that pH-driven ligand release involves ionization changes in several titratable residues associated with CD and EF loop apposition and occlusion of the calyx.     

Improved affinity of engineered streptavidin for the Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the binding site

The Strep-tag II is a nine-amino acid peptide that was developed as an affinity tool for the purification of corresponding fusion proteins on streptavidin columns. The peptide recognizes the same pocket of streptavidin where the natural ligand is normally bound so that biotin or its chemical derivatives can be used for competitive elution. We report here the crystal structures of the streptavidin mutants '1' and '2,' which had been engineered for 10-fold higher affinity towards the Strep-tag II. Both streptavidin mutants carry mutations at positions 44, 45, and 47, that is, in a flexible loop region close to the binding site. The crystal structures of the two apo-proteins and their complexes with the Strep-tag II peptide were refined at resolutions below 2 A. Both in the presence and absence of the peptide, the lid-like loop next to the ligand pocket--comprising residues 45 through 52--adopts an 'open' conformation in all four subunits within the asymmetric unit. The same loop was previously described to be disordered in the wild-type apo-streptavidin and to close over the pocket upon complexation of the natural ligand biotin. Our findings suggest that stabilization of the 'open' loop conformation in the absence of a ligand abolishes the need for conformational rearrangement prior to the docking of the voluminous peptide. Because no direct contacts between the flexible part of the loop and the peptide ligand were detected, it seems likely that the higher affinity of the two streptavidin mutants for the Strep-tag II is caused by a predominantly entropic mechanism.     

Structures of Escherichia coli branched-chain amino acid aminotransferase and its complexes with 4-methylvalerate and 2-methylleucine: induced fit and substrate recognition of the enzyme.

The following three-dimensional structures of three forms of Escherichia coli branched-chain amino acid aminotransferase (eBCAT) have been determined by the X-ray diffraction method: the unliganded pyridoxal 5'-phosphate (PLP) form at a 2.1 A resolution, and the two complexes with the substrate analogues, 4-methylvalerate (4-MeVA) as the Michaelis complex model and 2-methylleucine (2-MeLeu) as the external aldimine model at 2.4 A resolution. The enzyme is a trimer of dimers, and each subunit consists of small and large domains, and the interdomain loop. The active site is formed by the residues at the domain interface and those from two loops of the other subunit of the dimer unit, and binds one PLP with its re-face directed toward the protein side. Upon binding of a substrate, Arg40 changes its side-chain direction to interact with the interdomain loop, and the loop, which is disordered in the unliganded form, shows its ordered structure on the active-site cavity, interacts with the hydrophobic side chain of the substrate, and shields it from the solvent region. The substrate binds to the active-site pocket with its alpha-hydrogen toward the protein side, its side-chain on the side of O3 of PLP, and its alpha-carboxylate on the side of the phosphate group of PLP. The hydrophobic side-chain of the substrate is recognized by Phe36, Trp126, Tyr129, Tyr164, Tyr31*, and Val109*. The alpha-carboxylate of the substrate binds to the unique site constructed by three polar groups (two main-chain NH groups of the beta-turn at Thr257 and Ala258 and the hydroxy group of Tyr95) which are activated by the access of Arg40 to the main-chain C=O group of the beta-turn and the coordination of Arg97 to the hydroxy group. Since Arg40 is the only residue that significantly changes its side-chain conformation and directly interacts with the interdomain loop and the beta-turn, the residue plays important roles in the induced fit of the interdomain loop and the alpha-carboxylate recognition of the substrate.