Article pubs.acs.org/jmc
Protonation State and Hydration of Bisphosphonate Bound to Farnesyl Pyrophosphate Synthase Takeshi Yokoyama,*,† Mineyuki Mizuguchi,† Andreas Ostermann,‡ Katsuhiro Kusaka,§ Nobuo Niimura,§ Tabias E. Schrader,∥ and Ichiro Tanaka§,⊥ †
Faculty of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0914, Japan Heinz Maier-Leibnitz Zentrum (MLZ), Technische Universität München, Lichtenbergstrasse 1, 85748 Garching, Germany § Frontier Research Center for Applied Atomic Sciences, Ibaraki University, 162-1 Shirakata, Tokai, Ibaraki 319-1106, Japan ∥ Jülich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Forschungszentrum Jülich GmbH, Lichtenbergstr. 1, 85747 Garching, Germany ⊥ College of Engineering, Ibaraki University, Naka-Narusawa 4-12-1, Hitachi, Ibaraki 316-8511, Japan
Downloaded by CENTRAL MICHIGAN UNIV on September 15, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/acs.jmedchem.5b01147
‡
S Supporting Information *
ABSTRACT: Farnesyl pyrophosphate synthase (FPPS) catalyzes the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate to FPP and is known to be a molecular target of osteoporosis drugs, such as risedronate (RIS), which is a nitrogen-containing bisphosphonate. The protonation states and hydration structure of RIS bound to FPPS were determined by neutron protein crystallography, which allows direct visualization of hydrogens and deuteriums. The structure analysis revealed that the phosphate groups of RIS were fully deprotonated with the abnormally decreased pKa, and that the roles of E93 and D264 consisted of canceling the extra negative charges upon the binding of ligands. Collectively, our neutron structures provided insights into the physicochemical properties during the bisphosphonate binding event.
■
INTRODUCTION Nitrogen-containing bisphosphonates (N-BPs), such as zoledronate (ZOL) and risedronate (RIS, Figure 1a), are a class of drugs that are used clinically for the treatment of bone diseases such as osteoporosis,1 Paget’s disease,2 and tumor associated osteolysis.3 N-BPs suppress excessive bone resorption by inhibiting farnesyl pyrophosphate synthase (FPPS) in osteo-
clasts. FPPS, a homodimeric and a key regulatory enzyme in the mevalonate pathway, catalyzes the elongation of dimethylallyl pyrophosphate (DMAPP) to geranyl pyrophosphate (GPP) and then to farnesyl pyrophosphate (FPP) by condensing isopentenyl pyrophosphate (IPP, Figure 1b,c). Inhibition of FPPS prevents the synthesis of FPP and other downstream products that are required for the post-translational prenylation of small GTPases, such as Ras, Rho, and Rab family proteins, which are necessary for the function of osteoclasts.4,5 RIS is a member of the N-BPs, which are pyrophosphate analogues in which the oxygen bridge is replaced by a carbon atom. RIS can associate/dissociate six hydrogen atoms (four POH groups, one α-OH (attached to the central carbon), and one amino group). The previous X-ray crystal structures of FPPS have revealed that a FPPS protein possesses the two individual binding sites of DMAPP/GPP and IPP, and RIS binds to the DMAPP/GPP binding site and is coordinated via Mg2+ ions with the aspartate-rich motif of the protein.6,7 It has also been reported that RIS is hydrated by a number of water molecules in the binding pocket. However, the networks of hydrogen bonds are quite complex and it is difficult to predict the protonation states of Asp and Glu that form hydrogen
Figure 1. Chemical structures of FPPS ligands: (a) risedronate; (b) isopentenyl pyrophosphate. (c) Scheme of the FPPS reactions, showing the chemical structures of DMAPP, GPP, and FPP. © XXXX American Chemical Society
Received: July 21, 2015
A
DOI: 10.1021/acs.jmedchem.5b01147 J. Med. Chem. XXXX, XXX, XXX−XXX
Downloaded by CENTRAL MICHIGAN UNIV on September 15, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/acs.jmedchem.5b01147
Journal of Medicinal Chemistry
Article
bonds with water molecules based on the X-ray structures solved at moderate resolution (e.g., 2 Å). The binding of the negatively charged ligands, such as RIS and IPP, should provide the protein with an extra negative charge and cause a change in the protonation states of acidic/basic residues or a structural change.8 The binding affinities of bisphosphonates for hydroxylapatite of bone and FPPS are important in the mechanism of action, and these values are affected by the pKa values of the bisphosphonates. A strong correlation has been found between the ζ potentials of hydroxyapatite under the BPs and the pKa values of their side chains.9 It is also known that the positive charge of the BP side chain and the acidity of the phosphate groups are important for the inhibitory potency.10,11 Therefore, compounds for which the pKa is optimized for both bone and FPPS affinities would be more effective drugs against osteoporosis. Neutron crystallography is a suitable method for the direct visualization of hydrogen and deuterium atoms; that is, it can provide the direction of water molecules and protonation states of amino acid residues at a moderate resolution such as 2.5 Å. Herein, we present the neutron crystal structures of FPPSRIS-Mg2+ (FPPS-RIS) ternary and FPPS-RIS-Mg2+-IPP (FPPSRIS-IPP) quaternary complexes at 2.4 Å resolution. The structural analysis provided the protonation states and the hydration structure of RIS bound to FPPS, and the structural comparison between them revealed the mechanisms of the charge balancing of FPPS upon the ligand binding event.
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RESULTS AND DISCUSSION Overall Structure. Neutron diffraction data of FPPS-RIS and FPPS-RIS-IPP were collected at the BIODIFF diffractometer at the Forschungs-Neutronenquelle Heinz MaierLeibnitz research reactor (FRM II) in Germany and the BL03 (IBARAKI biological crystal diffractometer, iBIX) installed at the pulsed neutron source of the Material and Life Science Experimental Facility (MLF) at the Japan Proton Accelerator Research Complex (J-PARC), respectively. Complementary Xray diffraction data were collected at beamlines NE3A and NW12A at the Photon Factory Advanced Ring (Tsukuba, Japan). The neutron structures of H/D-exchanged FPPS-RIS and FPPS-RIS-IPP were refined by X/N joint refinement at 2.4 Å resolution (Supporting Information Table S1). The asymmetric unit contained an FPPS monomer (343 amino acid residues), and a functional FPPS dimer could be obtained by the crystallographic symmetry operation (Figure 2a). RIS bound to the DMAPP/GPP binding site with 3 Mg2+ ions, and the resulting complex was hydrated by 20 water molecules, and IPP bound to the pocket next to the DMAPP/GPP binding site as previously reported.12 The overall structure of FPPS-RIS was almost identical to that of FPPS-RIS-IPP, with the rmsd value being 0.15 Å. The protonation state of amino acid residues and the directions of hydroxyl groups and water molecules were determined by the difference Fourier maps omitting the hydrogen/deuterium atoms. The refined FPPS-RIS structure included 106 water molecules, and 61 of them were determined to be DOD. Two water clusters were found. The water molecules that bound to the RIS binding pocket were classified into cluster 1, and those that formed a hydrogen bond network next to the binding site of the cluster 1 water molecules were classified into cluster 2 (Figures 2a and 3a and Supporting Information Table S2).
Figure 2. Overall structure of FPPS. (a) Neutron structure of the FPPS-RIS-IPP dimer. FPPS is drawn as a cartoon model. The H/Dunexchanged region (4σ) but not in the FPPS-RIS structure, indicating that E93 was protonated upon the binding of IPP (Figure 5). In the IPP binding region, three water molecules (w547, w558, and w559) were conserved between FPPS-RIS and FPPS-RISIPP (rmsd = 0.7 Å), but the hydrogen-bond patterns were different because of the IPP binding and the protonation of E93. Whereas w547 donated the D2 atom in the hydrogen bond with E93 in FPPS-RIS, E93 donated the Dε2 atom in the hydrogen bond in FPPS-RIS-IPP. The OAH atom of the
structure) of RIS. In addition, w510 and w506, which were coordinated to the Mg2+ ions, hydrogen-bonded to Q240-Oε1 and D244-Oδ1, respectively. These water molecules are likely to strengthen the binding of RIS to FPPS. The pKa value of pyridine of RIS has been shown to be 6.1, and thus, the nitrogen atom of the pyridine ring of RIS would be unprotonated in aqueous solution.13 Therefore, FPPS must select a protonated RIS or increase its pKa during the RIS binding event. Zoledronate (ZOL), an imidazole bisphosphonate, exhibits slightly higher inhibitory potency than RIS,12,14 but the binding mode of ZOL has been shown to be similar to that of RIS. The higher inhibitory potency of ZOL might be accounted for by the higher pKa of the imidazole ring compared to the pyridine ring. Intriguingly, no neutron scattering length density around the phosphate groups of RIS was found in the difference Fourier map omitting the deuterium atoms, suggesting that the phosphate groups were fully deprotonated (Figure 4a). The phosphate groups were thoroughly surrounded by the hydrogen-bond donors, Mg2+ ions, and basic amino acid residues such as R112, K200, and K257, and consequently, it was sterically impossible for the hydrogens to bind to the phosphate groups (Supporting Information Figure S2). In addition, the low B-factors of the phosphate groups (7.8 Å2) proved that the phosphate groups were statically well ordered (Supporting Information Figure S1b and Figure S1c). These observations suggested that the pKa of the phosphate group was abnormally decreased upon binding to FPPS, since its pKa for the first protonation step is 12.0 and almost all the RIS should be partly protonated at physiological pH (crystallization condition: pD = 5.0).13 Although nine acidic amino acid residues were located close to RIS, they interacted indirectly with RIS. Mg2+ ions and basic amino acid residues, such as R112, K200, and K257, directly interacted with RIS so that the interactions with these cations are likely to decrease its pKa (Figure 4a). In addition, C
DOI: 10.1021/acs.jmedchem.5b01147 J. Med. Chem. XXXX, XXX, XXX−XXX
Downloaded by CENTRAL MICHIGAN UNIV on September 15, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/acs.jmedchem.5b01147
Journal of Medicinal Chemistry
Article
Figure 4. Structure around the phosphate groups with neutron scattering density map. The omit difference neutron maps around the phosphate groups of RIS (a) and the pyrophosphate group of IPP (b) are shown. In the case of IPP, all deuterium atoms were omitted for the map calculation. Omit maps are contoured at (4.0−7.0)σ. The Fourier peaks clearly belonged to the water molecules or amino acid residues but not to the phosphate groups. The yellow dashed lines represent hydrogen bonds.
pyrophosphate group was 5.7 Å distant from the Oε2 of E93. The pKa of E93 should be raised because the proximity of IPP and the water rearrangement would be necessary for the protonation of E93. E93 is widely conserved in prenyltransferases from various species, including FPPS, geranylgeranyl pyrophosphate synthase (GGPPS), and hexaprenyl pyrophosphate synthase (HexPPs), as are various aspartic acids (D103, D104, D107, D243, D244, and D247) that are located at the substrate binding region.6,16 Although several mutant experiments have revealed that these aspartic acids were important for the binding of the substrate or the catalytic reaction, there is no information on E93.17−20 The electric net charges around RIS and IPP were calculated and were found to be 0 in the structures of both FPPS-RIS and FPPS-RIS-IPP (Supporting Information Table S3). Thus, it was strongly suggested that the physiological role of E93 would be to balance the charge against the binding of IPP. It should be noted that K57 in the FPPSRIS structure was not included in these calculations. While the side chain of K57 was hydrogen bonded to the phosphate group of IPP in the FPPS-RIS-IPP structure, it was completely
disordered in the FPPS-RIS structure and was located at the molecular surface. The static permittivity of water is decreased 80-fold relative to that in a vacuum or hydrophobic environment. When calculating the net charge, it was thus considered reasonable to exclude the residue interacting with bulk water. Hydration of Risedronate. Two clusters of water molecules were found. Cluster 1 was composed of 20 water molecules (w501−508 and w522−533), and these water molecules hydrated RIS bound to FPPS (Figure 3a). The mean B value of cluster 1 was 14.2 Å2, which is much less than the mean B value of the asymmetric unit (20.5 Å2), indicating that the water molecules of cluster 1 were statically well ordered. Since the FPPS-RIS complex contains such a large hydrated structure inside the protein, it would be an appropriate model for statistical investigation of the nature of hydrogen bonds. The number of hydrogen bonds formed by cluster 1 was 43 in accordance with the criteria of D···O distance of
Protonation State and Hydration of Bisphosphonate Bound to Farnesyl Pyrophosphate Synthase Takeshi Yokoyama,*,† Mineyuki Mizuguchi,† Andreas Ostermann,‡ Katsuhiro Kusaka,§ Nobuo Niimura,§ Tabias E. Schrader,∥ and Ichiro Tanaka§,⊥ †
Faculty of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0914, Japan Heinz Maier-Leibnitz Zentrum (MLZ), Technische Universität München, Lichtenbergstrasse 1, 85748 Garching, Germany § Frontier Research Center for Applied Atomic Sciences, Ibaraki University, 162-1 Shirakata, Tokai, Ibaraki 319-1106, Japan ∥ Jülich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Forschungszentrum Jülich GmbH, Lichtenbergstr. 1, 85747 Garching, Germany ⊥ College of Engineering, Ibaraki University, Naka-Narusawa 4-12-1, Hitachi, Ibaraki 316-8511, Japan
Downloaded by CENTRAL MICHIGAN UNIV on September 15, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/acs.jmedchem.5b01147
‡
S Supporting Information *
ABSTRACT: Farnesyl pyrophosphate synthase (FPPS) catalyzes the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate to FPP and is known to be a molecular target of osteoporosis drugs, such as risedronate (RIS), which is a nitrogen-containing bisphosphonate. The protonation states and hydration structure of RIS bound to FPPS were determined by neutron protein crystallography, which allows direct visualization of hydrogens and deuteriums. The structure analysis revealed that the phosphate groups of RIS were fully deprotonated with the abnormally decreased pKa, and that the roles of E93 and D264 consisted of canceling the extra negative charges upon the binding of ligands. Collectively, our neutron structures provided insights into the physicochemical properties during the bisphosphonate binding event.
■
INTRODUCTION Nitrogen-containing bisphosphonates (N-BPs), such as zoledronate (ZOL) and risedronate (RIS, Figure 1a), are a class of drugs that are used clinically for the treatment of bone diseases such as osteoporosis,1 Paget’s disease,2 and tumor associated osteolysis.3 N-BPs suppress excessive bone resorption by inhibiting farnesyl pyrophosphate synthase (FPPS) in osteo-
clasts. FPPS, a homodimeric and a key regulatory enzyme in the mevalonate pathway, catalyzes the elongation of dimethylallyl pyrophosphate (DMAPP) to geranyl pyrophosphate (GPP) and then to farnesyl pyrophosphate (FPP) by condensing isopentenyl pyrophosphate (IPP, Figure 1b,c). Inhibition of FPPS prevents the synthesis of FPP and other downstream products that are required for the post-translational prenylation of small GTPases, such as Ras, Rho, and Rab family proteins, which are necessary for the function of osteoclasts.4,5 RIS is a member of the N-BPs, which are pyrophosphate analogues in which the oxygen bridge is replaced by a carbon atom. RIS can associate/dissociate six hydrogen atoms (four POH groups, one α-OH (attached to the central carbon), and one amino group). The previous X-ray crystal structures of FPPS have revealed that a FPPS protein possesses the two individual binding sites of DMAPP/GPP and IPP, and RIS binds to the DMAPP/GPP binding site and is coordinated via Mg2+ ions with the aspartate-rich motif of the protein.6,7 It has also been reported that RIS is hydrated by a number of water molecules in the binding pocket. However, the networks of hydrogen bonds are quite complex and it is difficult to predict the protonation states of Asp and Glu that form hydrogen
Figure 1. Chemical structures of FPPS ligands: (a) risedronate; (b) isopentenyl pyrophosphate. (c) Scheme of the FPPS reactions, showing the chemical structures of DMAPP, GPP, and FPP. © XXXX American Chemical Society
Received: July 21, 2015
A
DOI: 10.1021/acs.jmedchem.5b01147 J. Med. Chem. XXXX, XXX, XXX−XXX
Downloaded by CENTRAL MICHIGAN UNIV on September 15, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/acs.jmedchem.5b01147
Journal of Medicinal Chemistry
Article
bonds with water molecules based on the X-ray structures solved at moderate resolution (e.g., 2 Å). The binding of the negatively charged ligands, such as RIS and IPP, should provide the protein with an extra negative charge and cause a change in the protonation states of acidic/basic residues or a structural change.8 The binding affinities of bisphosphonates for hydroxylapatite of bone and FPPS are important in the mechanism of action, and these values are affected by the pKa values of the bisphosphonates. A strong correlation has been found between the ζ potentials of hydroxyapatite under the BPs and the pKa values of their side chains.9 It is also known that the positive charge of the BP side chain and the acidity of the phosphate groups are important for the inhibitory potency.10,11 Therefore, compounds for which the pKa is optimized for both bone and FPPS affinities would be more effective drugs against osteoporosis. Neutron crystallography is a suitable method for the direct visualization of hydrogen and deuterium atoms; that is, it can provide the direction of water molecules and protonation states of amino acid residues at a moderate resolution such as 2.5 Å. Herein, we present the neutron crystal structures of FPPSRIS-Mg2+ (FPPS-RIS) ternary and FPPS-RIS-Mg2+-IPP (FPPSRIS-IPP) quaternary complexes at 2.4 Å resolution. The structural analysis provided the protonation states and the hydration structure of RIS bound to FPPS, and the structural comparison between them revealed the mechanisms of the charge balancing of FPPS upon the ligand binding event.
■
RESULTS AND DISCUSSION Overall Structure. Neutron diffraction data of FPPS-RIS and FPPS-RIS-IPP were collected at the BIODIFF diffractometer at the Forschungs-Neutronenquelle Heinz MaierLeibnitz research reactor (FRM II) in Germany and the BL03 (IBARAKI biological crystal diffractometer, iBIX) installed at the pulsed neutron source of the Material and Life Science Experimental Facility (MLF) at the Japan Proton Accelerator Research Complex (J-PARC), respectively. Complementary Xray diffraction data were collected at beamlines NE3A and NW12A at the Photon Factory Advanced Ring (Tsukuba, Japan). The neutron structures of H/D-exchanged FPPS-RIS and FPPS-RIS-IPP were refined by X/N joint refinement at 2.4 Å resolution (Supporting Information Table S1). The asymmetric unit contained an FPPS monomer (343 amino acid residues), and a functional FPPS dimer could be obtained by the crystallographic symmetry operation (Figure 2a). RIS bound to the DMAPP/GPP binding site with 3 Mg2+ ions, and the resulting complex was hydrated by 20 water molecules, and IPP bound to the pocket next to the DMAPP/GPP binding site as previously reported.12 The overall structure of FPPS-RIS was almost identical to that of FPPS-RIS-IPP, with the rmsd value being 0.15 Å. The protonation state of amino acid residues and the directions of hydroxyl groups and water molecules were determined by the difference Fourier maps omitting the hydrogen/deuterium atoms. The refined FPPS-RIS structure included 106 water molecules, and 61 of them were determined to be DOD. Two water clusters were found. The water molecules that bound to the RIS binding pocket were classified into cluster 1, and those that formed a hydrogen bond network next to the binding site of the cluster 1 water molecules were classified into cluster 2 (Figures 2a and 3a and Supporting Information Table S2).
Figure 2. Overall structure of FPPS. (a) Neutron structure of the FPPS-RIS-IPP dimer. FPPS is drawn as a cartoon model. The H/Dunexchanged region (4σ) but not in the FPPS-RIS structure, indicating that E93 was protonated upon the binding of IPP (Figure 5). In the IPP binding region, three water molecules (w547, w558, and w559) were conserved between FPPS-RIS and FPPS-RISIPP (rmsd = 0.7 Å), but the hydrogen-bond patterns were different because of the IPP binding and the protonation of E93. Whereas w547 donated the D2 atom in the hydrogen bond with E93 in FPPS-RIS, E93 donated the Dε2 atom in the hydrogen bond in FPPS-RIS-IPP. The OAH atom of the
structure) of RIS. In addition, w510 and w506, which were coordinated to the Mg2+ ions, hydrogen-bonded to Q240-Oε1 and D244-Oδ1, respectively. These water molecules are likely to strengthen the binding of RIS to FPPS. The pKa value of pyridine of RIS has been shown to be 6.1, and thus, the nitrogen atom of the pyridine ring of RIS would be unprotonated in aqueous solution.13 Therefore, FPPS must select a protonated RIS or increase its pKa during the RIS binding event. Zoledronate (ZOL), an imidazole bisphosphonate, exhibits slightly higher inhibitory potency than RIS,12,14 but the binding mode of ZOL has been shown to be similar to that of RIS. The higher inhibitory potency of ZOL might be accounted for by the higher pKa of the imidazole ring compared to the pyridine ring. Intriguingly, no neutron scattering length density around the phosphate groups of RIS was found in the difference Fourier map omitting the deuterium atoms, suggesting that the phosphate groups were fully deprotonated (Figure 4a). The phosphate groups were thoroughly surrounded by the hydrogen-bond donors, Mg2+ ions, and basic amino acid residues such as R112, K200, and K257, and consequently, it was sterically impossible for the hydrogens to bind to the phosphate groups (Supporting Information Figure S2). In addition, the low B-factors of the phosphate groups (7.8 Å2) proved that the phosphate groups were statically well ordered (Supporting Information Figure S1b and Figure S1c). These observations suggested that the pKa of the phosphate group was abnormally decreased upon binding to FPPS, since its pKa for the first protonation step is 12.0 and almost all the RIS should be partly protonated at physiological pH (crystallization condition: pD = 5.0).13 Although nine acidic amino acid residues were located close to RIS, they interacted indirectly with RIS. Mg2+ ions and basic amino acid residues, such as R112, K200, and K257, directly interacted with RIS so that the interactions with these cations are likely to decrease its pKa (Figure 4a). In addition, C
DOI: 10.1021/acs.jmedchem.5b01147 J. Med. Chem. XXXX, XXX, XXX−XXX
Downloaded by CENTRAL MICHIGAN UNIV on September 15, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/acs.jmedchem.5b01147
Journal of Medicinal Chemistry
Article
Figure 4. Structure around the phosphate groups with neutron scattering density map. The omit difference neutron maps around the phosphate groups of RIS (a) and the pyrophosphate group of IPP (b) are shown. In the case of IPP, all deuterium atoms were omitted for the map calculation. Omit maps are contoured at (4.0−7.0)σ. The Fourier peaks clearly belonged to the water molecules or amino acid residues but not to the phosphate groups. The yellow dashed lines represent hydrogen bonds.
pyrophosphate group was 5.7 Å distant from the Oε2 of E93. The pKa of E93 should be raised because the proximity of IPP and the water rearrangement would be necessary for the protonation of E93. E93 is widely conserved in prenyltransferases from various species, including FPPS, geranylgeranyl pyrophosphate synthase (GGPPS), and hexaprenyl pyrophosphate synthase (HexPPs), as are various aspartic acids (D103, D104, D107, D243, D244, and D247) that are located at the substrate binding region.6,16 Although several mutant experiments have revealed that these aspartic acids were important for the binding of the substrate or the catalytic reaction, there is no information on E93.17−20 The electric net charges around RIS and IPP were calculated and were found to be 0 in the structures of both FPPS-RIS and FPPS-RIS-IPP (Supporting Information Table S3). Thus, it was strongly suggested that the physiological role of E93 would be to balance the charge against the binding of IPP. It should be noted that K57 in the FPPSRIS structure was not included in these calculations. While the side chain of K57 was hydrogen bonded to the phosphate group of IPP in the FPPS-RIS-IPP structure, it was completely
disordered in the FPPS-RIS structure and was located at the molecular surface. The static permittivity of water is decreased 80-fold relative to that in a vacuum or hydrophobic environment. When calculating the net charge, it was thus considered reasonable to exclude the residue interacting with bulk water. Hydration of Risedronate. Two clusters of water molecules were found. Cluster 1 was composed of 20 water molecules (w501−508 and w522−533), and these water molecules hydrated RIS bound to FPPS (Figure 3a). The mean B value of cluster 1 was 14.2 Å2, which is much less than the mean B value of the asymmetric unit (20.5 Å2), indicating that the water molecules of cluster 1 were statically well ordered. Since the FPPS-RIS complex contains such a large hydrated structure inside the protein, it would be an appropriate model for statistical investigation of the nature of hydrogen bonds. The number of hydrogen bonds formed by cluster 1 was 43 in accordance with the criteria of D···O distance of
