Structural basis of protein stability at poly extreme crystal structure of amya at 1 6 a resolution 4

CHAPTER CRYSTAL STRUCTURE OF AmyA AT VERY HIGH SALT 4.1 INTRODUCTION Biophysical experiments suggest that AmyA retains the overall fold over the entire salinity range tested However, the molecular mechanism by which AmyA adapts to different salt concentration and confers thermal stability is not clearly understood To know the mechanism at the atomic level we crystallized the protein from a solution containing a very high NaCl concentration (4.7 M) Apart from this high salt 1.8 M ammonium sulfate was used as a precipitant To the best of our knowledge this is the highest salt concentration at which a protein has been crystallized According to our biophysical studies we have found that AmyA maintains the overall fold even at a very high salt concentration The crystal structure that we have determined at very high salt reveals that the subtle differences in the side chain conformation and excess metal ion binding play a major role in stabilizing AmyA at very high salinity 4.2 MATERIAL AND METHODS 4.2.1 Crystallization of AmyA at very high salt concentration The purified AmyA protein was dialyzed against 50 mM Tris buffer (pH 7.5) containing 4.7 M NaCl and was concentrated up to 5.5 mg ml-1 Crystallization attempts using the hanging drop and sitting drop methods failed to produce crystals as vapor diffusion must have occurred from the reservoir to the crystallization drop because of the high salt concentration of the protein solution To avoid such a diffusion effect, the micorbatch-under-oil method (Chayen, et al, 1992) was used 68 Crystals formed in a buffer containing 0.01 M cobalt chloride hexahydrate, 0.1 M MES (pH 6.5) and 1.8 M ammonium Sulphate in addition to the protein solution having 4.7 M NaCl For cryo-protection, crystals were soak-transferred in crystallization solutions containing increasing amounts of glycerol to a final glycerol concentration of 25% X-ray diffraction data for the high salt AmyA crystal were collected at -173 °C from flash-cooled crystals using synchrotron radiation at NSLS, Brookhaven (beamline X12B) All diffraction images were processed with HKL2000 (Otwinowski and Minor, 1997) The crystal belongs to the monoclinic P21 space group and contains one molecule per asymmetric unit Figure 4.1 Crystal picture of AmyA formed at high salt concentration The structure was solved by the molecular replacement method, by using the atomic coordinates of the low salt AmyA structure Subsequently, the final model was built by using ARP/wARP (Perrakis et a., 1999) All stages of crystallographic refinement made use of CNS (Brunger et a., 1998) The calcium and chloride ions 69 were identified from the 2|Fo|-|Fc| and |Fo|-|Fc| σ weighted maps In the difference Fourier map, positive peaks with density greater than σ values were considered as ions Different plausible ions were placed on a trial and error basis and refined The B factors of these ions were compared with those of the surrounding main chain and side chain atoms Correct coordination geometry was also verified to confirm and accept correct metal ions Furthermore, any possible misassumption of calcium for sodium was cross-checked When we placed sodium ions instead of calcium ions we observed very low B-factor values and strong unaccounted positive density in the difference Fourier map near sodium which clearly justified and confirmed that these were not sodium ions The crystallographic parameters, data collection and refinement details are given in Tables 4.1 and 4.2 Molecular figures were generated in PYMOL (http://pymol.org) Different atomic contacts were calculated using the WHAT IF program (Rodriguez et al, 1998) 70 Table 4.1 Crystal parameters and data collection statistics of hAmyA Space group P21 a = 40.52 Unit cell dimensions (Å, º) b = 74.74 c = 78.05 β = 104.67 Wavelength (Å) 0.99 Resolution of data (Å) 50-1.83 (1.9-1.83) No of measured reflections 75,848 No of unique reflections 38,904 Redundancy 1.95 Completeness (%) 97.4 (93.2) Mean I / σ(I) 11.5 (2.1) 0.050 (0.36) Rmerge -Numbers in parentheses are for the outermost shell of data 4.3 Rmerge =ΣhklΣi |Ii(h k l) – | / ΣhklΣi Ii(h k l)] RESULTS 4.3.1 Overview of AmyA structure at very high salt Crystals in the P21 space group contain one monomer per asymmetric unit The overall fold of the AmyA structure in high salt (hereafter called as hAmyA) (Fig 4.1) remains the same as that of the AmyA structure at low salt (hereafter called as lAmyA) The hAmyA and lAmyA structures can be superimposed with an RMSD of 0.78 Å for the backbone Cα atoms of the entire chain lengths (Fig 4.2) 71 Table 4.2 Refinement statistics of hAmyA Resolution range (Å) 10-1.83 (1.83-1.94) |F|/σ(|F|) >0 Protein atoms 4,025 Water molecules 546 Calcium Chloride Rwork (%) 20.0 (26.7) Rfree (%) 24.7 (30.4) Reflections (working/test) 36,650 / 3,669 RMS deviations from ideal stereochemistry Bond lengths (Å) 0.006 Bond angles (º) 1.20 Mean B factors Protein (Å ) Water (Å ) Ions (Å ) 16.34 27.52 28.52 - -1 Rwork =∑hkl||Fo(hkl)| – |Fc(hkl)|| / ∑hkl|Fo(hkl)| 2Rfree is equivalent to Rwork but is calculated for randomly chosen 10% of reflections omitted from the refinement process However, there are marked differences in the side chain conformations of charged residues The side chains of the entire chain lengths are superimposed with an RMSD of 1.34 Å The notable difference that we find in the main chain is in the active site (residues 220-333) conformation with a main chain RMSD of 1.31 Å The active site 72 of hAmyA is better superimposed with that of other known α-amylase structures than that of lAmyA In lAmyA, the main chain and side chain of the catalytic residue Asp224 protrude outside from the active site where the nucleophilic reaction is proposed to occur (Fig 4.3) This could be one of the reasons why AmyA requires a high concentration of salt for optimal activity and shows reduced activity at low salt concentration The highly disordered loop, residues 159-173, has similar characteristics, like high temperature factors and multiple occupancies, in both hAmyA and lAmyA Among calcium binding loops, the loop of residues 64-73 shows differences in both side chains and the main chain (Fig 4.4) However, in both the lAmyA and hAmyA structures the loop binds to calcium ion Figure 4.2 A ribbon diagram of the hAmyA molecule The three domains are represented in different colors and the calcium and chloride ions are shown as violet and cyan colored spheres, respectively 73 Figure 4.3 Stereo image of the superimposition of the structures of lAmyA (blue) and hAmyA (green); calcium ions, corresponding to hAmyA and lAmyA, are shown as spheres in the respective protein colors; the chloride ion of hAmyA is represented as a cyan sphere Figure 4.4 Active site comparisons A stereo view of the active site cleft and comparison of the key catalytic residues of lAmyA (green) and hAmyA (purple) The conserved catalytic residues are labeled 74 Figure 4.5 A stereo view of a calcium binding loop shown in the stick model and the calcium ion is shown as a sphere The lAmyA and hAmyA are in blue and green color respectively To understand the structural features that are responsible for stability, particularly at high ionic strength, hAmyA was compared against lAmyA The hAmyA structure forms a total of 37 salt bridges while lAmyA has only 33 Also, 10 of the hAmyA salt bridges are at different locations when compared to the salt bridges in lAmyA This difference is also to some extent due to the effect of crystal lattice formation as lAmyA and hAmyA crystals are formed in different space groups Three salt bridges are influenced in both lAmyA and hAmyA primarily because of the residues’ involvement in crystal lattice formation as they form intermolecular interactions In both crystallization conditions no additional calcium was added However, hAmyA binds to a total of calcium ions and one chloride ion whereas lAmyA contains only two calcium ions and no chloride ion is found The number of potential hydrogen bonds was determined by the WHAT IF program suit Both the structures have similar number of H-bonds, 1335 in hAmyA and 1330 for lAmyA However, their positions and networks have differences The structural differences between hAmyA and lAmyA described above, like increased amount of metal ion binding, salt bridge and their network, are nearly equivalent to the differences 75 between thermophilic and mesophilic protein structures The differences between thermophilic and mesophilic proteins are due to specific substitution of amino acids at solvent exposed regions, whereas here the sequence of AmyA is identical in the two salt conditions and the differences occur mainly due to the side chain dynamics 4.3.2 Surface property of hAmyA The distribution of positively and negatively charged residues in the hAmyA structure has a similar trend to that at 0.5M NaCl, Table 4.3.The hAmyA structure has a more electro positive surface potential (Fig 4.5) compared to lAmyA, mainly due to the increased number of calcium ions bound and differences in the side chain conformation of polar residues These surface analyses clearly indicate the unique surface nature of AmyA when compared to other halophilic proteins Table 4.3 Exposure of charged residues on AmyA surface Crystal structure of AmyA No of Surface from residues charge Asp/Glu Arg/Lys His Halothermothrix orenii Out In Out In Out In Out In At Low salt 488 +0.5 -8.5 48 23 45 11 7 At High salt 488 +3.0 -11.0 45 26 45 11 The assignment whether a residue is exposed to the outer surface (out) or the inner surface (in) was based on the crystal structures and the solvent accessibility of each residue, as calculated using the program WHAT IF The estimate of overall surface charges was calculated by assuming fully ionized states for Asp, Glu, Lys, Arg and +0.5 charge for His 76 A B Figure 4.6 The surface property of AmyA (A) The electrostatic surface potential of lAmyA and (B) hAmyA The electrostatic drawings were produced using the program GRASP (Nicholls et a., 1991) Surface colors represent the potential from -10 kBT-1 (red) to +10 kBT-1 (blue) 4.3.3 Calcium binding At high salt concentration the availability of water molecules for protein hydration is less and also sodium salts are more soluble than calcium salts So a trace of calcium ions present in water binds to this low affinity calcium pocket in the protein which would normally be hydrated at low salt concentration This could be the physical explanation for the excess binding of calcium to AmyA at high salt The 77 Calcium No Interacting atoms Distance in Å ASN 443-OD1 2.55 GLY 445-O 2.68 WAT-13 2.70 WAT-83 2.70 WAT-140 2.70 Calcium Calcium 79 Calcium Calcium Figure 4.7 A stereo view of calcium binding loops with electron density The 2Fo–Fc electron density omit maps are drawn at the 1.3 σ contour level Side chains of the amino acids that make coordination bonds with calcium ions are shown as sticks and the calcium and water molecules are shown as magenta and red spheres, respectively 4.3.4 Novel calcium and chloride binding Binding of calcium ions to α-amylases is known to stabilize their structures highly The hAmyA structure binds to five calcium ions and one chloride ion Two of the calcium binding sites are novel when compared to other known α-amylase 80 structures The novel calcium and chloride binding sites are present in the vicinity of the active site and interact with each other, coordinated by two helices (Fig 4.7) This kind of ionic interaction between calcium and chloride ions has not been observed in other amylases However, the conserved calcium ion that is usually present at the interface of domains A and B in most of the thermophilic α-amylases is absent in AmyA Comparisons of metal ion binding in different mesophilic and thermophilic amylases (Table 4.6) clearly indicate that AmyA binds to larger number of metal ions similarly to other thermophiles Table 4.5 The coordinating atoms of the calcium-chloride with distances No Interacting atoms Distance in Å HIS 306-NE2 2.83 ASP 266-OE1 2.63 ASP 266-OE2 2.91 Chloride 701-Cl 2.80 WAT-456 2.93 WAT-457 2.75 81 Figure 4.8 A stereo representation of the calcium and chloride binding loop with electron density The 2Fo–Fc electron density omit maps are drawn at the 1.3 σ contour level Side chains of the amino acids that make coordination bonds with calcium ions are shown as sticks and the calcium and water molecules are shown as magenta and red spheres, respectively Table 4.6 The number of metal ions in AmyA and other homologous amylase structures PDB id Organism Extremophilic Optimum Number of nature temperature metal ions °C 1WML- Halothermothrix Halo-thermophilic 65 hAmyA orenii 1WZA- Halothermothrix Halo-thermophilic 65 lAmyA orenii 1UOK Bacillus cereus Mesophilic 35 1M53 Klebsiella sp, LX13 Mesophilic 35 1SMA Thermus sp Thermophilic 65 1JI2 Thermoactinomyces Thermophilic 65 vulgaris 1LWH Thermotoga maritama Thermophilic 80 1MWO Pyrococus woesei Hyperthermophilic 100 82 4.3.5 Structural determinants of thermal stability of AmyA To determine the conserved features of thermophilic proteins that are present in AmyA, the AmyA structure was compared with other known thermophilic and mesophilic α-amylase structures Ion-pair networks are thought to be a major determinant for protein stability at high temperatures The hAmyA structure contains ion-pair networks and each network involves at least residues interconnecting various secondary structure elements (Fig 4.8 A-D) The salt bridges with the distances and their network are listed in Table 4.7 The networks a and b that further involve in hydrogen bonding with water molecules WAT33 and WAT110 reside at the center of the barrel structure, where the active site is present Network c is present in most thermophilic amylases AmyA’s closest structural homolog oligo-1,6glucosidase (mesophilic, PDB ID: IUOK) contains only two such networks involving and residues each Comparison of ion-pair network in different homologous amylases is given in Table 4.8 Figure 4.9a Figure 4.9b 83 Figure 4.9c Figure 4.9d Figure 4.9 Ion-pair networks of AmyA (a,b,c and d) A close up stereo view of the ion-pair networks Secondary structures are represented as ribbons and the residues that are involved in ion-pair networks are shown as ball-and-sticks Positive and negative groups are shown in blue and red color, respectively 84 Table 4.7 Salt bridge networks in hAmyA Network No Participating residues Distance in Å ASP-277-OD2 - LYS- 30-NZ 3.23 ASP-277-OD1 - ARG-246-NH2 3.05 ASP-277-OD2 - ARG-246-NH1 2.96 ASP-277-OD2 - ARG-246-NH2 3.25 GLU-250-OE1 - ARG-246-NH2 3.52 Network No Participating residues Distance in Å GLU- 36-OE2 - HIS-329-ND1 2.66 ASP-330-OD1 - HIS-329-NE2 3.70 ASP-330-OD2 - HIS-329-NE2 3.13 ASP-330-OD1 - ARG-379-NH2 2.84 Network No Participating residues Distance in Å ASP-128-OD1 - ARG-222-NH1 3.52 ASP-128-OD2 - ARG-222-NH1 2.97 ASP-224-OD1 - ARG-222-NH1 2.65 ASP-224-OD2 - ARG-222-NH1 2.85 GLU-260-OE1 - ARG-222-NH2 3.60 GLU-260-OE2 - ARG-222-NH2 3.65 Network No Participating residues Distance in Å GLU-440-OE1 - ARG-452-NH1 2.62 ASP-459-OD1 - ARG-452-NH2 3.19 ASP-459-OD2 - ARG-452-NH2 3.51 GLU-513-OE1 - ARG-458-NH2 2.91 GLU-513-OE2 - ARG-458-NH2 3.59 85 The number of ion-pair networks were calculated for lAmyA, hAmyA and other homologous mesophilic and thermophilic α-amylases The networks contain at least residues each In the calculation, the maximum distance between the two groups involved in forming salt bridges was set to 3.6 Å Furthermore, aromatic amino acid interactions are known to be one of the determinants of thermal stability in thermophilic proteins (Kannan and Vishveshwara, 2000; Serrano et a., 1991) A pair of aromatic interactions contributes between -0.6 and -1.3 kcal.mol-1 to protein stability (Serrano et a., 1991) AmyA sequence contains about 15% excess of aromatics amino acids when compared to its mesophilic counterparts In the AmyA structure, there are several clusters of closely interacting aromatic amino acids, two of which involve 29 and 13 residues, respectively (Fig 4.9) These structural elements, metal ion binding, ion-pair network and aromatic clusters, acting at both the protein surface and the core of AmyA might contribute collectively and significantly to the thermal stability of AmyA 86 Table 4.8 The number of ion pair networks of AmyA and other homologous amylases PDB id Organism Extremophilic Optimum Number Nature Temperature of °C ion-pair Networks 1WML- Halothermothrix orenii Halo-thermophilic 65 Halothermothrix orenii Halo-thermophilic 65 1UOK Bacillus cereus Mesophilic 35 1M53 Klebsiella sp, LX13 Mesophilic 35 1SMA Thermus sp Thermophilic 65 1JI2 Thermoactinomyces Thermophilic 65 hAmyA 1WZAlAmyA vulgaris 1LWH Thermotoga maritama Thermophilic 80 1MWO Pyrococus woesei Hyperthermophilic 100 87 Figure 4.10 Aromatic clusters of AmyA The aromatic amino acids are represented as dot surface in the AmyA cartoon diagram The two large aromatic clusters of AmyA are shown in pink and cyan colors and the third cluster in the C-terminal domain is shown in blue color 4.3.6 Structural determinants of halophilic stability of AmyA AmyA is the first halophilic α-amylase protein structure that has been solved to date We compared it with other halophilic protein structures Apart from the conserved acidic surface, there are other reported stabilizing structural elements in halophilic proteins like increased number of surface exposed salt bridges and binding of anions and cations that render protein stability These characteristic features are also observed in AmyA However, these characteristics are common to thermophilic proteins also The other unique and important factor in halophilic proteins is the binding of hydrated ions on the protein surface (Bieger et al, 2003) In AmyA calcium ion Ca5 is hexa-coordinated and binds to three water molecules, Fig 4.10 88 Figure 4.11 A novel calcium binding site contains three water molecules The 2Fo–Fc electron density map is drawn at the 1.5 σ contour level The side chains are shown as sticks and the calcium (magenta) and water (red) molecules are shown as spheres Each of the other calcium ions has at least one coordinated water molecule There are some positive peaks present in the difference Fourier map posing to be ions They have either lower sigma values than the assigned ions due to partial occupancy or they lack the coordination sphere around them Also, when declared as water, they show lower B-factors, compared to normal water molecules Thus these sites could be hydrated ions that need confirmation by some other experimental techniques This increased binding of hydrated ions at high salt concentration could be important to keep the protein hydrated and soluble This property of AmyA could complement the solvation effect that an acidic surface produces in other halophilic proteins 89 4.3.7 Solvation of AmyA and its implication Both the structures are equally well hydrated, Fig 4.11 However there are differences in the position of water molecule binding around the protein surface In the lAmyA and hAmyA structures 551 and 546 water molecules have been located, respectively This indicates that water binding capacity of AmyA does not get affected in high salt solution despite the lack of excess acidic residues A B Figure 4.12 Overview of the solvation sphere of AmyA (A) lAmyA and (B) hAmyA The surfaces of AmyA molecules are shown in grey color and the water molecules are shown as red spheres 90 CHAPTER DISCUSSION We have solved the first crystal structure of a halo- and thermophilic protein at 1.6 Å Both the low and high salt structures provide a molecular insight into the structural features that contribute to the stability of AmyA in two extreme conditions Unlike typical halophilic proteins, AmyA and other H orenii proteins need to adapt to a broad salinity range in addition to high temperatures because the Tunisian salt lakes have large seasonal fluctuations in their salt concentration (Najia et a., 2001) Halophilic proteins in general unfold and have low thermal stability at low salt concentrations as a consequence of the repulsive force exerted by the excess of acidic residues on their surfaces From the AmyA crystal structures we propose that the reduction in the number of negatively charged residues is adequately compensated by the presence of an equal number of positively charged residues, and the consequent formation of ion-pairs and their networks allow AmyA, and possibly other H orenii proteins, to have high thermal stability in a broad salinity range AmyA is the first known protein that is stable in saturated salt solutions but lacks an acidic surface Our finding does not necessarily contradict the ‘acidic surface nature’ of halophilic proteins but provides evidence of another novel stabilization mechanism AmyA lacks the halophilic signature of acidic surface but has thermophilic structural elements such as aromatic amino acid clustering, salt bridge network and metal ion binding One might hence ask whether these thermophilic adaptations are sufficient to impart halophilic stability? Results from various studies on the effect of salt on the stability of various thermophilic proteins suggest that it is very unlikely The effect of salt on the activity and stability of thermophilic proteins appears to vary with the individual protein (Vieille and Zeikus, 2001) However, these proteins have 91 rarely been tested in molar level salt concentration to completely rule out this possibility Also, it is evident from the AmyA structure that it also lacks some of the thermophilic signatures such as reduction in loop structure AmyA contains extended loop structures similar to mesophilic amylases In AmyA most of these extended loops binds to hydrated metal ions, which are considered essential for halophilic proteins It is also necessary to address to what extent halophilic adaptations contribute to thermophilic stability Interestingly, halophilic proteins show higher thermal stability and optimum activity at higher temperature when compared to their mesophilic counterparts However, the increase in thermal stability is observed only in the presence of high salt concentration These results suggest that proteins require unique structural features to be stable at the poly-extremes of high temperature and salinity Structure-based sequence alignment suggests that AmyA might have evolved from a common ancestral bacterial amylase gene by specific amino acid substitutions at the solvent accessible surface Comparisons of hAmyA and lAmyA structures provide a fascinating example of the way in which the change in side chain conformations and protein dynamics bring in differences in the structural elements and their stabilizing effects at different environments It has been reported that the effect of salt on surface exposed electrostatic interactions is destabilizing but the effect is low at high ionic strength (Mueller et al, 2000) This could be the reason why AmyA surprisingly has more close ionic interactions at high ionic strength than at low ionic strength The two AmyA structures thus illustrate the clever strategy used by the protein to handle two extreme conditions simultaneously Our biophysical studies, combined with structural studies, suggest that AmyA retains the same overall fold in the entire salinity and in the complete absence of salt it 92 forms poly-dispersed oligomers Functional implications of this reversible oligomer formation in the absence of salt could be to increase thermal stability as it loses its thermal stability at low salt concentration in the monomeric state Thus to maintain the same structure at low salt condition and at higher temperature, AmyA forms favorable oligomers This is the first time this kind of stabilization mechanism has been observed at low salt for a halophilic protein Stability of proteins at both high temperatures and high salt allows for interesting biotechnological and industrial applications involving processes performed at both chemical and physical extremes Comparison with other thermophilic amylases suggests that the AmyA function and stability could be improved further for biotechnological applications by mutations at specific sites Introducing calcium binding sites in domain B may increase the thermal stability of AmyA as most of the thermophilic proteins are stabilized by calcium binding at the interface of domains A and B Also, the AmyA structure contains structural features common to both thermophilic and halophilic proteins which suggest that proteins can be engineered to function at multiple extreme conditions by carefully combining the structural properties of proteins from different extremophiles However, more proteins need to be crystallized and studied before we can reach at general conclusions on any specific structural feature Our work here provides the first few insights into the structural basis of poly-extreme stability of AmyA More work involving mutational analysis and theoretical calculations would be necessary to develop a quantitative understanding of the different stabilizing features It is our hope and belief that this study certainly sets the stage for a long lasting future investigation of proteins at polyextreme conditions 93 ... To understand the structural features that are responsible for stability, particularly at high ionic strength, hAmyA was compared against lAmyA The hAmyA structure forms a total of 37 salt bridges... The active site 72 of hAmyA is better superimposed with that of other known α-amylase structures than that of lAmyA In lAmyA, the main chain and side chain of the catalytic residue Asp2 24 protrude... space group contain one monomer per asymmetric unit The overall fold of the AmyA structure in high salt (hereafter called as hAmyA) (Fig 4 .1) remains the same as that of the AmyA structure at