19F NMR studies of the D-galactose chemosensory receptor. 1. Sugar binding yields a global structural change.

The Escherichia coli D-galactose and D-glucose receptor is an aqueous sugar-binding protein and the first component in the distinct chemosensory and transport pathways for these sugars. Activation of the receptor occurs when the sugar binds and induces a conformational change, which in turn enables docking to specific membrane proteins. Only the structure of the activated receptor containing bound D-glucose is known. To investigate the sugar-induced structural change, we have used 19F NMR to probe 12 sites widely distributed in the receptor molecule. Five sites are tryptophan positions probed by incorporation of 5-fluorotryptophan; the resulting 19F NMR resonances were assigned by site-directed mutagenesis. The other seven sites are phenylalanine positions probed by incorporation of 3-fluorophenylalanine. Sugar binding to the substrate binding cleft was observed to trigger a global structural change detected via 19F NMR frequency shifts at 10 of the 12 labeled sites. Two of the altered sites lie in the substrate binding cleft in van der Waals contact with the bound sugar molecule. The other eight altered sites, specifically two tryptophans and six phenylalanines distributed equally between the two receptor domains, are distant from the cleft and therefore experience allosteric structural changes upon sugar binding. The results are consistent with a model in which multiple secondary structural elements, known to extend between the substrate cleft and the protein surface, undergo shifts in their average positions upon sugar binding to the cleft. Such structural coupling provides a mechanism by which sugar binding to the substrate cleft can cause structural changes at one or more docking sites on the receptor surface.     

Large-scale domain movements and hydration structure changes in the active-site cleft of unligated glutamate dehydrogenase from Thermococcus profundus studied by cryogenic X-ray crystal structure analysis and small-angle X-ray scattering

Here we describe the large-scale domain movements and hydration structure changes in the active-site cleft of unligated glutamate dehydrogenase. Glutamate dehydrogenase from Thermococcus profundus is composed of six identical subunits of M(r) 46K, each with two distinct domains of roughly equal size separated by a large active-site cleft. The enzyme in the unligated state was crystallized so that one hexamer occupied a crystallographic asymmetric unit, and the crystal structure of the hexamer was solved and refined at a resolution of 2.25 A with a crystallographic R-factor of 0.190. In that structure, the six subunits displayed significant conformational variations with respect to the orientations of the two domains. The variation was most likely explained as a hinge-bending motion caused by small changes in the main chain torsion angle of the residue composing a loop connecting the two domains. Small-angle X-ray scattering profiles both at 293 and 338 K suggested that the apparent molecular size of the hexamer was slightly larger in solution than in the crystalline state. These results led us to the conclusion that (i) the spontaneous domain motion was the property of the enzyme in solution, (ii) the domain motion was trapped in the crystallization process through different modes of crystal contacts, and (iii) the magnitude of the motion in solution was greater than that observed in the crystal structure. The present cryogenic diffraction experiment enabled us to identify 1931 hydration water molecules around the hexamer. The hydration structures around the subunits exhibited significant changes in accord with the degree of the domain movement. In particular, the hydration water molecules in the active-site cleft were rearranged markedly through migrations between specific hydration sites in coupling strongly with the domain movement. We discussed the cooperative dynamics between the domain motion and the hydration structure changes in the active site of the enzyme. The present study provides the first example of a visualized hydration structure varying transiently with the dynamic movements of enzymes and may form a new concept of the dynamics of multidomain enzymes in solution.     

Functional dynamics of the hydrophobic cleft in the N-domain of calmodulin

Molecular dynamics studies of the N-domain (amino acids 1-77; CaM(1-77)) of Ca2+-loaded calmodulin (CaM) show that a solvent exposed hydrophobic cleft in the crystal structure of CaM exhibits transitions from an exposed (open) to a buried (closed) state over a time scale of nanoseconds. As a consequence of burying the hydrophobic cleft, the R(g) of the protein is reduced by 1.5 A. Based on this prediction, x-ray scattering experiments were conducted on this domain over a range of concentrations. Models built from the scattering data show that the R(g) and general shape is consistent with the simulation studies of CaM(1-77). Based on these observations we postulate a model in which the conformation of CaM fluctuates between two different states that expose and bury this hydrophobic cleft. In aqueous solution the closed state dominates the population, while in the presence of peptides, the open state dominates. This inherent flexibility of CaM may be the key to its versatility in recognizing structurally distinct peptide sequences. This model conflicts with the currently accepted hypothesis based on observations in the crystal structure, where upon Ca2+ binding the hydrophobic cleft is exposed to solvent. We postulate that crystal packing forces stabilize the protein conformation toward the open configuration.     

Features of F(1)-ATPase catalytic and noncatalytic sites revealed by fluorescence lifetimes and acrylamide quenching of specifically inserted tryptophan residues

Catalytic and noncatalytic nucleotide sites of the F(1) sector of ATP synthase were characterized by tryptophan fluorescence techniques. Seven Trp residues inserted in varied microenvironments in the catalytic sites, and one in the noncatalytic sites, were studied in mutant F(1) enzymes which were otherwise devoid of Trp. Parameters measured were fluorescence lifetimes and dynamic and static quenching by acrylamide in the absence or presence of nucleotide. The results indicated that the solution structures of the mutant enzymes were consistent with reported crystal structures. In enzyme with three empty noncatalytic sites, all sites were relatively inaccessible to acrylamide, indicating a closed conformation. In contrast, when the three catalytic sites were empty, they were relatively and equally accessible to acrylamide, indicating an open conformation. This was the case in the presence or absence of Mg(2+). Residue beta-Trp-331 has been extensively used previously to determine nucleotide binding parameters in F(1). Results here showed that in betaY331W mutant F(1), each of the three beta-Trp-331 residues has an unusually long fluorescence lifetime, confirming that each contributes equally to the overall fluorescence signal.     

Solution X-ray scattering evidence for agonist- and antagonist-induced modulation of cleft closure in a glutamate receptor ligand-binding domain

Agonist-induced conformational changes in the ligand-binding domains (LBD) of glutamate receptor ion channels provide the driving force for molecular rearrangements that mediate channel opening and subsequent desensitization. The resulting regulated transmembrane ion fluxes form the basis for most excitatory neuronal signaling in the brain. Crystallographic analysis of the GluR2 LBD core has revealed a ligand-binding cleft located between two lobes. Channel antagonists stabilize an open cleft, whereas agonists stabilize a closed cleft. The crystal structure of the apo form is similar to the antagonist-bound, open state. To understand the conformational behavior of the LBD in the absence of crystal lattice constraints, and thus better to appreciate the thermodynamic constraints on ligand binding, we have undertaken a solution x-ray scattering study using two different constructs encoding either the core or an extended LBD. In agreement with the GluR2 crystal structures, the LBD is more compact in the presence of agonist than it is in the presence of antagonist. However, the time-averaged conformation of the ligand-free core in solution is intermediate between the open, antagonist-bound state and the closed, agonist-bound state, suggesting a conformational equilibrium. Addition of peptide moieties that connect the core domain to the other functional domains in each channel subunit appears to constrain the conformational equilibrium in favor of the open state.     

Actin-induced closure of the actin-binding cleft of smooth muscle myosin

The putative actin-binding interface of myosin is separated by a large cleft that extends into the base of the nucleotide binding pocket, suggesting that it may be important for mediating the nucleotide-dependent changes in the affinity for myosin on actin. We have genetically engineered a truncated version of smooth muscle myosin containing the motor domain and the essential light chain-binding region (MDE), with a single tryptophan residue at position 425 (F425W-MDE) in the actin-binding cleft. Steady-state fluorescence of F425W-MDE demonstrates that Trp-425 is in a more solvent-exposed conformation in the presence of MgATP than in the presence of MgADP or absence of nucleotide, consistent with closure of the actin-binding cleft in the strongly bound states of MgATPase cycle for myosin. Transient kinetic experiments demonstrate a direct correlation between the rates of strong actin binding and the conformation of Trp-425 in the actin-binding cleft, and suggest the existence of a novel conformation of myosin not previously seen in solution or by x-ray crystallography. Thus, these results directly demonstrate that: 1) the conformation of the actin-binding cleft mediates the affinity of myosin for actin in a nucleotide-dependent manner, and 2) actin induces conformational changes in myosin required to generate force and motion during muscle contraction.     

19F NMR studies of the D-galactose chemosensory receptor. 2. Ca(II) binding yields a local structural change.

The Escherichia coli D-galactose and D-glucose receptor possesses a Ca(II)-binding site closely related in structure and metal-binding characteristics to the eukaryotic EF-hand sites. Only the structure of the Ca(II)-occupied site is known. To investigate the structural change triggered by Ca(II) and Sr(II) binding, we have used 19F NMR to probe five 5-fluorotryptophan (5F-Trp) and seven 3-fluorophenylalanine (3F-Phe) positions in the structure, extending the approach described in the preceding article. Of particular interest were two 5F-Trp residues near the N terminus of the Ca(II) site at positions 127 and 133. Substitution of the larger Sr(II) for Ca(II) triggered 19F NMR frequency shifts of the 5F-Trp127 and -133 resonances, indicating a detectable structural change in the Ca(II) site. In contrast, the three 5F-Trp resonances from distant regions of the structure exhibited no detectable frequency shifts. When the metal was removed from the Ca(II) site, the 5F-Trp127 and -133 frequencies shifted to a new value similar to that observed for free 5F-Trp in aqueous solvent, and this new frequency was a function of the H2O to D2O ratio, indicating that the residues had become solvent exposed. Metal removal yielded small or undetectable frequency shifts for the three distant 5F-Trp resonances and for four of the five resolved 3F-Phe resonances. The allosteric coupling of the metal and sugar binding sites was observed to be slight: depletion of metal ions was observed to reduce the D-galactose affinity of the receptor by 2-fold. Together the results indicate that the structural changes in the Ca(II) site are primarily localized in the region of the site. Removal of the metal ion from the site exposes the nearby 5F-Trp127 and -133 residues to the solvent, suggesting that the empty site has a more open structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chondrodystrophic mice with coincidental agnathia: evidence for the tongue obstruction hypothesis in cleft palate

Mice homozygous for either of two mutations, chondrodysplasia (cho) or cartilage matrix deficiency (cmd), have short-limbed chondrodystrophy. This phenotype includes retrognathia, relative macroglossia, and cleft palate. It has been postulated that the cleft palate in these mice is the result of tongue obstruction during palatogenesis. Agnathia associated with microglossia is an independent spontaneously occurring defect in the strains bearing these mutations. The coincidental occurrence of agnathia-microglossia with chondrodystrophy lends itself to the study of the mechanism of cleft palate formation. We examined approximate midsagittal histological sections of normal and chondrodystrophic newborn mice, both with and without agnathia. Mandibular measurements and examinations of palate closure and tongue structure were made from photographic prints. Typical chondrodystrophic mutants with cleft palates had a mean mandibular length that was 66% of normal and a tongue that appeared large relative to the shortened mandible. Chondrodystrophic mutants with agnathia and microglossia had a mean mandibular length that was further reduced to 30% of normal, yet had a closed palate. We also observed two nonagnathic chondrodystrophic mutants that had slightly decreased mandibular lengths, microglossia, and closed palates. These observations suggest that tongue obstruction during palatogenesis is the pathogenetic mechanism of cleft palate in chondrodystrophic mice. A similar tongue obstruction hypothesis has been proposed as the mechanism of cleft palate formation in the human Pierre Robin sequence, which consists of retrognathia, glossoptosis, and cleft palate. This mechanistic hypothesis has been challenged, but our findings support the tongue obstruction hypothesis in the Robin cleft.     

Primary correction of the unilateral cleft lip nose: a 15-year experience

This paper reviews a 15-year personal experience based on 400 unilateral cleft nasal deformities that were reconstructed using a method that repositions the alar cartilage by freeing it from the skin and lining and shifts it to a new position. The rotation-advancement lip procedure facilitates the exposure and approach to the nasal reconstruction. The nasal soft tissues are transected from the skeletal base, reshaped, repositioned, and secured by using temporary stent sutures that readapt the alar cartilage, skin, and lining. The nasal floor is closed and the ala base is positioned to match the normal side. Good subsequent growth with maintenance of the reconstruction has been noted in this series. The repair does not directly expose or suture the alar cartilage. Improvement in the cleft nasal deformity is noted in 80 percent of the cases. Twenty percent require additional techniques to achieve the desired symmetry. This method has been used by the author as his primary unilateral cleft nasal repair and has been taught to residents and fellows under his direction with good results. This technique eliminates the severe cleft nasal deformity seen in many secondary cases.     

Energetics of the cleft closing transition and the role of electrostatic interactions in conformational rearrangements of the glutamate receptor ligand binding domain

The ionotropic glutamate receptors are localized in the pre- and postsynaptic membrane of neurons in the brain. Activation by the principal excitatory neurotransmitter glutamate allows the ligand binding domain to change conformation, communicating opening of the channel for ion conduction. The free energy of the GluR2 S1S2 ligand binding domain (S1S2) closure transition was computed using a combination of thermodynamic integration and umbrella sampling modeling methods. A path that involves lowering the charge on E705 was chosen to clarify the role of this binding site residue. A continuum electrostatics approach in S1S2 is used to show E705, located in the ligand binding cleft, stabilizes the closed conformation of S1S2 via direct interactions with other protein residues, not through the ligand. In the closed conformation, in the absence of a ligand, S1S2 is somewhat more closed than what has been reported in X-ray structures. A semiopen conformation has been identified which is characterized by disruption of a single cross-cleft interaction and differs only slightly in energy from the fully closed S1S2. The fully open S1S2 conformation exhibits a wide energy well and shares structural similarity with the apo S1S2 crystal structure. Hybrid continuum electrostatics/MD calculations along the chosen closure transition pathway reveal solvation energies, and electrostatic interaction energies between two lobes of the protein increase the relative energetic difference between the open and closed conformational states. By analyzing the role of several cross-cleft contacts as well as other binding site residues, we demonstrate how S1S2 interactions facilitate formation of the closed conformation of the GluR2 ligand binding domain.     

Comparison of the periplasmic receptors for L-arabinose, D-glucose/D-galactose, and D-ribose. Structural and Functional Similarity.

The primary sequence of the receptor for L-arabinose or Ara-binding protein (ABP) composed of 306 residues is very different from the D-glucose/D-galactose-binding protein (GGBP) which consists of 309 residues. Nevertheless, superimpositioning of the well-refined high resolution structures of ABP in complex with D-galactose and the GGBP in complex with D-glucose shows very similar structures; 220 of the residues (or about 70%) have a root mean square deviation of 2.0 A. From the superpositioning, nine pairs of continuous segments (consisting of 8-51 residues), mainly alpha-helices and beta-strands that form the core of the two lobes of the bilobate proteins were found to exhibit strong sequence homology. The equivalenced structures and aligned sequences show that many of the polar, as well as aromatic residues, in the sugar-binding sites located in the cleft between the two lobes are highly conserved. Surprisingly, however, the exact mode of binding of the D-galactose in ABP is totally different from that of the D-glucose in GGBP. Using the structurally aligned sequences of the ABP and GGBP as a template, we have matched the sequence of the ribose-binding protein (RBP) which consists of 271 residues with the ABP/GGBP pair. Although the nine aligned segments of all three proteins show little sequence identity, they have significant homology. Four additional segments of RBP were matched only with GGBP, leading to the alignment of about 90% of the RBP sequence with the GGBP sequence. Many of the conserved residues in the binding sites of ABP and GGBP matched with similar residues in RBP. Additional observations indicate that the GGBP/RBP pair is more closely related than the ABP/RBP or ABP/GGBP pair. All three binding proteins, which may have diverged from a common ancestor, serve as primary receptors for bacterial high affinity active transport systems. Moreover, GGBP and RBP, but not ABP, also act as receptors for chemotaxis. An exposed site located in one domain, which includes Gly74, for interacting with the trg transmembrane signal transducer that is involved in triggering chemotaxis has been located in the structure of GGBP (Vyas, N.K., Vyas, M.N., and Quiocho, F.A. (1988) Science 242, 1290-1295). Whereas the site is absent in the structure of ABP, it is strongly predicted to be present in RBP which shares the same trg transducer with GGBP. The knowledge-based alignment of RBP further revealed two possible additional peripheral chemotactic sites that show high structural and sequence similarity between GGBP and RBP only. At least one of these sites, together with the one proven to exist in the other domain, could be used by the signal transducer with which both binding proteins interact in a way which the substrate-loaded "closed cleft" structure could be discriminated from the unliganded "open cleft" form by the transducer.     

Direct evidence for the quantum theory of the cell transformation by use of the nucleoproteide equivalents' staining

The separation of the nucleoproteide equivalents of human Fogh and Lund [FL] cells by means of sucrose gradient centrifugation devoted 5 separate distinguished fractions. A treatment of the cells by pancreatic RNase and/or extraction of RNA by hypertonic 0.9% sodium chloride solution eliminated the last 3 fractions. An increase of the acidification of the intercalating thiazine dyes by acetic acid at 2.9 less than or equal to pH less than or equal to 5.5 accumulated the complete diffuse dispersed nucleoproteide pool in the endoplasmic reticulum and nucleus gradually over formation of chromosomal aberrations and cluster into a heap of nucleoproteide crystals with empty cytoplasmic zones.     

Assessment of bilateral cleft lip nose deformity: a comparison of results as judged by cleft surgeons and laypersons

Reconstruction of bilateral cleft lip nose deformity is difficult and the outcome is inconsistent. This study was conducted to evaluate the gross outcome and the difference in the assessment of nasal appearance as judged by two groups of raters, cleft surgeons and laypersons. Sixty-four patients with bilateral cleft lip were selected for review. The patients' ages ranged from 5 to 30 years. All patients had undergone primary cleft lip repair and secondary nasal reconstruction, and had been followed for at least 6 months. One image for each patient, which included a digitized frontal, lateral, and worm's-eye view, was projected for evaluation by the raters. The raters included five cleft surgeons and five laypersons. A rating scheme was used in which a score of 3 was given for a good, close to normal nasal appearance, 2 for an average result that needed minor revision, and 1 for a poor result that needed major reconstruction. The scores were averaged for each patient in each group and for each group as a whole. The final outcome was judged as good, fair, or poor on the basis of the mean score for each patient. Statistical analysis was performed. The mean score for all patients was 2.08 as assessed by the laypersons and 2.18 as assessed by the cleft surgeon group. There was no statistically significant difference between the two groups. Comparisons on rating scores among different raters revealed a fair agreement on the ratings within each of the two groups. The results were found to be good in 29.7 percent, fair in 64.1 percent, and poor in 6.3 percent of patients when evaluated by the surgeons. When rated by the laypersons, the nasal appearance was found to be good in 26.6 percent, fair in 60.9 percent, and poor in 12.5 percent of patients. This difference in distribution between the two groups was not statistically significant. When comparing the results given by the two groups of assessors, there was agreement on the nasal appearance in 65.6 percent of patients, and a difference in grading in the rest. For the patients who received different grading, the surgeons rated them one grade higher in 63.6 percent and one grade lower in 36.4 percent. There was no difference in grading between any of the evaluators that reflected a two-grade discrepancy in evaluation of results. This study shows that the surgical outcome of bilateral cleft lip nose deformity repair, at the authors' institution, is less than optimal. When assessing bilateral cleft lip nose appearance, the judgment of results by cleft surgeons was similar to that of the laypersons. However, different rating of results existed within each of the two groups, supporting the importance of clearly assessing patient/parent expectations and defining realistic surgical goals.     

Structural basis for cyclodextrin recognition by Thermoactinomyces vulgaris cyclo/maltodextrin-binding protein

The crystal structure of a Thermoactinomyces vulgaris cyclo/maltodextrin-binding protein (TvuCMBP) complexed with gamma-cyclodextrin has been determined. Like Escherichia coli maltodextrin-binding protein (EcoMBP) and other bacterial sugar-binding proteins, TvuCMBP consists of two domains, an N- and a C-domain, both of which are composed of a central beta-sheet surrounded by alpha-helices; the domains are joined by a hinge region containing three segments. gamma-Cyclodextrin is located at a cleft formed by the two domains. A common functional conformational change has been reported in this protein family, which involves switching from an open form to a sugar-transporter bindable form, designated a closed form. The TvuCMBP-gamma-cyclodextrin complex structurally resembles the closed form of EcoMBP, indicating that TvuCMBP complexed with gamma-cyclodextrin adopts the closed form. The fluorescence measurements also showed that the affinities of TvuCMBP for cyclodextrins were almost equal to those for maltooligosaccharides. Despite having similar folds, the sugar-binding site of the N-domain part of TvuCMBP and other bacterial sugar-binding proteins are strikingly different. In TvuCMBP, the side-chain of Leu59 protrudes from the N-domain part into the sugar-binding cleft and orients toward the central cavity of gamma-cyclodextrin, thus Leu59 appears to play the key role in binding. The cleft of the sugar-binding site of TvuCMBP is also wider than that of EcoMBP. These findings suggest that the sugar-binding site of the N-domain part and the wide cleft are critical in determining the specificity of TvuCMBP for gamma-cyclodextrin.     

Cleft palate: more genetic lessons from mice

Cleft palate occurred in high frequency (14%) in the F2 generation of the cross between two stocks of mice, LGG and SELH, neither of which produces more than 2% cleft palate. The cleft palate trait results from a new combination of alleles that is not present in either parental stock. The lack of cleft palate in the F2 generation after outcrosses of both parental stocks to other strains shows that this new combination of alleles has specific contributions from both parental strains, and also that there must be at least two loci involved. A deficiency of Mod-1 homozygotes in the SELH/LGG F2 adults suggests that one of the loci involved may be linked to Mod-1 and that the number of loci involved is few. Significantly more F2 males (19%) than females (9%) were affected with cleft palate. The data can be explained by a two-locus epistatic model with a dominant mutation (P) at one locus that causes cleft palate when not suppressed by or compensated for by a dominant allele (S) at a second locus. The parental stocks would be PPSS and ppss. In the F2 generation, the new combinations PPss and Ppss would express cleft palate, a total expected of 19%. Similar new combinations of alleles at two loci may explain some instances of high occurrence of cleft palate or other developmental threshold traits in previously unaffected human families.     

Lower incidence of nonsyndromic cleft lip with or without cleft palate in females: Is homocysteine a factor?

In India, as in other parts of the world, nonsyndromic cleft lip with or without cleft palate (NSCL±P) is a highly prevalent birth defect, its incidence in males being twice that in females. A case–control association study has been carried out with respect to homocysteine level and MTHFR C677T, A1298C and SLC19A1 (RFC1) G80A genotypes from an eastern Indian cohort to investigate whether Hcy and other Hcy-pathway genes also contribute to the risk level. While MTHFR 677T and SLC19A1 80G are individually and cumulatively risk factors, SLC19A1 80A appears to be protective against MTHFR 677T risk allele. Elevated Hcy associates with NSCL±P both in case mothers and cases. Significantly, this difference shows a gender bias: the level of elevation of Hcy in female cases is distinctly higher than in males, and more case females are hyperhomocyteinemic than the case males. It implies that compared with the males, higher level of Hcy is needed for NSCL±P to manifest in the females. We consider this as one of the possible factors why the incidence of this disorder in females is much lower than in males.     

Surgical treatment of the cleft foot

Between 1970 and 1997, we treated a total of 32 cleft feet in 21 patients (11 male and 10 female). We classified cases of cleft foot on the basis of the number of central ray deficiencies. Fourteen patients with 22 cleft feet were followed up for more than 1 year postoperatively (9 feet had no or one central ray deficiency, and 13 feet had two or three central ray deficiencies). The mean follow-up period was 8.8 years. The objective of this study was to evaluate the results of operative treatment of cleft foot. We evaluated the results of three methods: simple closure of the cleft, application of a double-pedicled flap, and insertion of a silicone block. Cosmetic complications, including widening of the foot, hypertrophic scarring, pigmentation of the grafted skin, and overlapping of the toes, were observed in patients with two or three central ray deficiencies. Few functional complications were observed: None of the patients experienced gait disturbances, although one patient complained of pain following walking. Roentgenography showed that the distance between the first and fifth metatarsals was 86 percent of that of the contralateral foot. When treating patients with no or one central ray deficiency, satisfactory results can be expected with simple closure of the cleft. However, in patients with two or three central ray deficiencies, it is difficult to obtain satisfactory results with simple closure of the cleft or application of a double-pedicled flap. Therefore, silicone block insertion to correct the defect is recommended when there is more than one central ray deficiency.     

Structural basis of compound recognition by adenosine deaminase

Structural snapshots corresponding to various states enable elucidation of the molecular recognition mechanism of enzymes. Adenosine deaminase has two distinct conformations, an open form and a closed form, although it has so far been unclear what factors influence adaptation of the alternative conformations. Herein, we have determined the first nonligated structure as an initial state, which was the open form, and have thereby rationally deduced the molecular recognition mechanism. Inspection of the active site in the nonligated and ligated states indicated that occupancy at one of the water-binding positions in the nonligated state was highly significant in determining alternate conformations. When this position is empty, subsequent movement of Phe65 toward the space induces the closed form. On the other hand, while occupied, the overall conformation remains in the open form. This structural understanding should greatly assist structure-oriented drug design and enable control of the enzymatic activity.