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

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 stabi

CHAPTER 4 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

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

were identified from the 2|Fo|-|Fc| and |Fo|-|Fc| σ weighted maps In the difference Fourier map, positive peaks with density greater than 9 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)

Table 4.1 Crystal parameters and data collection statistics of

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)

No of measured reflections 75,848

No of unique reflections 38,904

Redundancy 1.95

Table 4.2 Refinement statistics of hAmyA

-

- -

-1Rwork =∑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

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

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

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 5 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

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

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

Surface charge

Asp/Glu Arg/Lys His Crystal structure of AmyA

from

Halothermothrix orenii

No of residues

Out In Out In Out In Out In

At Low salt 488 +0.5 -8.5 48 23 45 11 7 7

At High salt 488 +3.0 -11.0 45 26 45 11 6 8

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

A

B

coordination atoms to the calcium ions are tabulated below in Table 4.4 The calcium binding loops are shown in Fig 4.6 along with electron density

Table 4.4 Coordinating atoms of calcium with distances

Calcium 4

Calcium 5

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

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

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

nature

Optimum temperature

°C

Number of metal ions

1WML-hAmyA

Halothermothrix orenii

1WZA-lAmyA

Halothermothrix orenii

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

4 ion-pair networks and each network involves at least 4 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 glucosidase (mesophilic, PDB ID: IUOK) contains only two such networks involving

oligo-1,6-4 and 3 residues each Comparison of ion-pair network in different homologous amylases is given in Table 4.8

Figure 4.9a

Figure 4.9b

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

Table 4.7 Salt bridge networks in hAmyA

3.23 3.05 2.96 3.25 3.52

2.66 3.70 3.13 2.84

3.52 2.97 2.65 2.85 3.60 3.65

2.62 3.19 3.51 2.91 3.59

The number of ion-pair networks were calculated for lAmyA, hAmyA and other homologous mesophilic and thermophilic α-amylases The networks contain at least 4 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

Table 4.8 The number of ion pair networks of AmyA and

other homologous amylases

Nature

Optimum Temperature

°C

Number

of ion-pair Networks

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