Structural basis of protein stability at poly extreme crystal structure of amya at 1 6 a resolution 2

Considerable effort has been made to date to understand the molecular mechanism of adaptation of proteins from thermophilic and halophilic organisms at the atomic level.. At the same tim

CHAPTER 2 CRYSTAL STRUCTURE DETERMINATION OF AMYA FROM

HALOTHERMOTHRIX ORENII

Some organisms can live in extreme conditions like temperatures that are close to the boiling point of water, pressures that are many hundred times that of atmospheric pressure and salinity that is orders of magnitude above typical physiological conditions These extremophiles use several cellular and structural adaptation mechanisms to be able to survive, actively grow and propagate in extreme environments It has been reported that macromolecules, especially proteins from extremophiles and mesophiles, have the same overall fold and molecular mechanism

for their function (Vieille et al, 1995; Russell et al, 1997; Bauer et al, 1998)

However, certain structural features make these proteins stable and optimally active under extreme conditions Considerable effort has been made to date to understand the molecular mechanism of adaptation of proteins from thermophilic and halophilic organisms at the atomic level

2.1.1 Thermophilic protein stability

With the exception of phylogenetic variations, what differentiates thermophilic and mesophilic enzymes is only the temperatureranges in which they are stable and active Otherwise, thermophilicand mesophilic enzymes are highly similar The sequences of homologous thermophilic and mesophilic proteins are typically

40 to 85% similar (Davies et al, 1993), their three-dimensional structures are

superimposable (Auerbach et al, 1998; Tahirov et al, 1998) and they have the same

catalytic mechanism (Bauer et al, 1998) Thermophilic proteins are the most widely

studied due to their extreme stability and ease of purification From these studies it appears that thermal stability is not determined by any single factor but the combination of several factors, each with a relatively small effect (Perutz and Raidt,

1975; Argos et al, 1979; Vogt et al, 1997; Jaenicke and Bohm, 1998; Shih and Kirsch,

1995) These factors include the following

2.1.1.1 Amino acid composition

The amino acid composition of a protein has long been thought to be correlated to its thermostability The first statistical analysis comparing amino acid compositions in mesophilic and thermophilic proteins indicated the trends toward substitutions such as Glycine to Alanine and Lysine to Arginine A higher Alanine content in thermophilic proteinswas supposed to reflect the fact that Alanine was the best helix-forming residue (Argos et al, 1979) As more experimental data

accumulate, in particular complete genome sequences, it is becoming obvious that

"traffic rules of thermophilic adaptation cannot be defined in terms of significant differences in the amino acid composition" (Bohm and Jaenicke, 1994) The comparison of residue contents in hyperthermophilic and mesophilic proteins based

on the genome sequences of mesophilic and hyperthermophilic organisms shows only minor trends

2.1.1.2 Hydrophobic interactions

The hydrophobic effect is considered to be a major driving force of protein folding Hydrophobicity drives the protein to a collapsed structure from which the native structure is defined by the contribution of all types of forces (Dill, 1990)

Thermophilic proteins normally have extensive hydrophobic interactions and reduced water accessiblehydrophobic surface area compared to their mesophilic counterparts

(Kannan and Vishveshwara, 2000; Serrano et al, 1991) A pair of aromatic interactions contributes between -0.6 and -1.3 kcal/mol to protein stability (Serrano et

al, 1991)

2.1.1.5 Ion-pair

Salt bridges are formed by spatially proximal pairs of oppositely charged residues in native protein structures Often salt-bridging residues are also close in the protein sequence and fall in the same secondary structural element, building block, autonomous folding unit, domain, or subunit, consistent with the hierarchical model for protein folding Salt bridges are rarely found across protein parts which are joined

by flexible hinges, a fact suggesting that salt bridges constrain flexibility and motion

While conventional chemical intuition expects that salt bridges contribute favorably to protein stability, recent computational and experimental evidence shows that salt bridges can be stabilizing or destabilizing Due to systemic protein flexibility, reflected in small-scale side-chain and backbone atom motions, salt bridges and their stabilities fluctuate in proteins At the same time, genomewide amino acid sequence composition, structural, and thermodynamic comparisons of thermophilic and mesophilic proteins indicate that specific interactions, such as salt bridges, may contribute significantly towards the thermophilic-mesophilic protein stability differential Ion pair networks are energetically more favorable than an equivalent number of isolated ion pairs because for eachnew pair the burial cost is cut in half: only one additional residuemust be desolvated and immobilized (Yip et al, 1995)

2.1.1.6 Metal binding

Metals have long been known to stabilize and activate enzymes In proteins, metal ions are coordinated, usually by lone pair electron donation from oxygen or nitrogen atoms Some thermophilic and hyperthermophilic enzymes have been reported that contain metal atoms that are not present in theirmesophilic homologs Experiments have shown that metal binding can contribute 6 - 9 kcal/mol to stability

2.1.1.7 Extrinsic parameters

While most pure hyperthermophilic enzymes are intrinsically very stable, some intracellular hyperthermophilic proteins get their high thermostability from intracellular environmental factors such as salts, high protein concentrations, coenzymes, substrates, activators, polyamines, or an extracellular environmental factorsuch aspressure

2.1.2 Halophilic protein stability

Halophiles (salt-lover) can be defined as microorganisms that require high salt

in a concentration range of 2 - 5 M to grow (Richard & Zaccai, 2000) In order to overcome the extreme osmotic pressure of these hyper saline environments, halophilic bacteria and eukaryotes accumulate mostly neutral organic compatible solutes and exclude most of the inorganic salts In contrast, halophilic archaea balance the external high salt concentration by intracellular accumulation of inorganic ions to concentrations that exceed that of the medium Therefore, all the cellular components

of the halophilic archaea must adapt to function at the extremely high intracellular salt concentration

Halophilic proteins require a minimum of 2 M salt concentration to be optimally active and stable At high salt concentrations, proteins are in general destabilized due to enhanced hydrophobic interactions Halophilic proteins have, therefore, evolved specific mechanisms that allow them to be both stable and soluble

in high salt concentration The adoptive mechanism of halophilic proteins has not been studied as extensively as thermophilic proteins due to the difficulty in purifying and crystallizing them at very high ionic strengths Halophilic enzymes are usually very unstable in low salt concentrations Since some of the important fractionation methods in protein chemistry, such as, electrophoresis and ion exchange chromatography, cannot be applied at high salt concentrations, the available fractionation methods for halophilic bacterial proteins are rather limited

In silico analyses of the genome sequence of halophilic organisms suggest that

proteins from these organisms have unique amino acid compositions They have at

least twice the number of acidic residues than basic residues (Fukuchi et al, 2003; Bieger et al, 2003) Structural insights gathered from the known halophilic crystal

structures suggest that the acidic surface and the associated negative electrostatic surface potential, is one of the major stabilizing forces and is a highly conserved

feature (Dym et al, 1995; Bieger et al, 2003), Fig 2.1

Figure 2.1 The electrostatic surface potential of malate dehydrogenase from

Haloarcula marismortui, a typical halophilic protein (PDB id: 1D3A) The

electrostatic drawings were produced using the program GRASP Surface colors represent the potential from -10 kBT-1 (red) to +10 kBT-1 (blue)

Since all soluble halophilic enzymes have a highly negative surface charge, once folded properly, their flexibility may be achieved by repulsive forces between closely placed charged residues The instability caused by the high surface charge density should be somehow balanced Otherwise, the polypeptide chain will unfold It was long believed that one of the roles of high salt concentration was to shield this high surface charge Indeed, classical electrostatic calculations using Poisson–Boltzmann equation (Elcock and McCammon, 1998) suggest that at pH 7.0 the

stability of halophilic proteins is decreased by 18.2 kcal/mol on lowering the salt concentration from 5 to 0.1 M

Using thermodynamic theories to analyze various biophysical measurements

(Bonnete et al, 1993) it was calculated that, in its native state at 4 M NaCl, halophilic

malate dehydrogenase (MDH) binds approximately 200 molecules of salt and almost

3000 molecules of water These values are significantly higher than those measured for non-halophilic proteins under the same condition and also higher than the number

of salt and water molecules bound in low salt solutions in which the halophilic enzyme is unfolded These findings are the basis for the ‘halophilic stabilization model’ for solutions in NaCl, KCl and MgCl2 (Zaccai, 1989) According to this model the tertiary and quaternary structures of native halophilic proteins co-ordinate hydrated salt ions on their surface at higher local concentrations than in the surrounding solution by specific interactions with the surface carboxyl groups Through the binding of hydrated salt ions, water molecules would be associated with the protein structure with different local salt concentrations depending on the hydrated interactions of the particular salt When the bulk salt concentration is reduced, salt will diffuse from the ‘quasi-crystalline’ protein-associated layer into the solvent bulk, destabilizing the protein surface and causing dissociation of the enzyme into its subunits and unfolding of the polypeptide chain According to this model, the stabilization is enthalpy driven The entropic penalty derived from the organization of the hydrated salt is compensated by the enthalpy of the binding of the hydrated salt to the surface carboxyl groups

This explanation for the role of salt in halophilic protein stabilization is challenged by two experimental results First, the high resolution three-dimensional

structure of Haloarcula marismortui ferredoxin (HmFd) demonstrates very clearly

that although the protein was crystallized from 3.8 M sodium–potassium phosphate, very few counter ions were found to be bound to the protein and when bound, they interact with the main-chain carbonyl oxygen and not with side-chain carboxylates (Frolow et al, 1996) Second, sub-millimolar concentrations of NADH can effectively replace the requirement for molar quantities of salt in the stabilization of halophilic malate dehydrogenase (MDH) Therefore, neutralization of surface charge by salt may not be required for protein stability (Irimia et al, 2003)

In addition, some of the thermophilic protein determinants like metal ion binding and salt bridge networks also play a role in stabilizing halophilic proteins

(Dym et al, 1995; Bieger et al, 2003)

2.1.3 Poly-extremophiles

Poly-extremophiles can be defined as the organisms that require more than one extreme condition for its optimal growth and survival For example, the organism

Thermosipho japonicus isolated from a deep-sea hydrothermal vent in the Okinawa

area, Japan requires high temperature and high pressure for its optimal growth (Takai

et al, 2000)

An interesting category among this involves the organisms that require both high temperatures and high salt concentrations Extremophilic microbes of this kind are rare in nature and those isolated so far are difficult to handle in routine laboratories To date only two such organisms have been reported, namely,

Cayol et al, 1994), both of which are members of the low G+C DNA-containing gram-positive phylum H orenii is a true halophilic and thermophilic anaerobic

bacterium that was isolated from the Tunisian salt lake in the Sahara desert It requires

2 M NaCl and 60 °C temperature for optimal growth but still shows significant

growth up to 4 M NaCl (Cayol et al, 1994) Halothermothrix orenii is a member of the family Haloanaerobiaceae, order Haloanaerobiales (Cayol et al, 1994), whereas

Thermohalobacter berrensis, a member of the order Clostridiales, grows readily at 70

°C in the presence of 15% NaCl (Cayol et al, 2000) These organisms drastically

differ from other extremophiles as they handle both physical and chemical extremes at the same time

The study of the molecular adaptation of proteins at more than one extreme i.e poly-extreme condition is very important in the understanding of the biology of these organisms A few specific questions may be posed in this halo- and thermophilic group of poly-extremophilies

1) Are the structural adaptations the same as those found in halophilic and thermophilic organisms or would it be a completely new set of adaptations? 2) In the former case, would these adaptations be a simple addition of structural features or are there complex interactions of these features?

3) What specific differences exist in the poly-extreme adaptations?

4) What is the impact of the structural adaptations on the function and mechanism of the proteins?

To address these questions we have undertaken a biophysical study of AmyA, a

secretory α-amylase (Mijts and Patel, 2002) from Halothermothrix orenii AmyA is

an endo-acting α-amylase and randomly cleaves the α-1,4-glycosidic linkages present

in starch and its constituent polysaccharides amylose and amylopectin (Mijts and Patel, 2002) AmyA is active in a broader salt concentration ranging between 0 and 5

M NaCl However, it has optimal activity at 2 M NaCl concentration and temperature

above 65 °C, similar to the optimal conditions for H orenii growth The obligatory

requirement of salt at molar levels and temperature above 60 °C makes AmyA a true halo-thermophilic enzyme

2.1.4 The α-amylases:

Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) are Endo-acting enzymes that catalyze the hydrolysis of alpha-1,4-glycosidic bonds in starch and related poly and oligosaccharides The α-amylase family comprises a group of enzymes with a variety of different specificities that all act on one type of substrate, being glucose residues linked through an α–1,1, 1,4, or 1,6 glycosidic bond The members of this family share a number of common characteristics but at least 21 different enzyme specificities are found within the family These differences in specificity are based not only on subtle differences within the active site of the enzymes but also on the differences within the overall architecture of the enzymes The α-amylase family can roughly be divided into two subgroups: the starch-hydrolyzing enzymes and the starch-modifying or transglycosylating enzymes The hydrolases and transferases that constitute the α-amylase family are multidomain proteins, but each has a catalytic domain in the form of a (β/α)8-barrel with the active site being at the C-terminal end of the barrel beta-strands Although the enzymes are believed to share the same catalytic acids and a common mechanism of action, they have been assigned to three separate families - 13, 70 and

77 - in the classification scheme for glycoside hydrolases and transferases that is based on amino acid sequence similarities

Figure 2.2 Different enzymes involved in the degradation of starch The

open ring structure symbolizes the reducing end of a polyglucose molecule

2.1.4.1 Domain Architecture of Amylase:

The enzymes are multidomain proteins, but share a common catalytic domain

in the form of a (β/α)8-barrel, i.e., a barrel of eight parallel β-strands surrounded by eight helices, the so-called domain A This structure has been demonstrated by X-ray crystallography in several enzymes of the α-amylase family, although in one instance only seven of the eight helices in the barrel fold are present In addition, studies of amino acid sequence similarities have led to the prediction that many other enzymes belong to this family and have a similar catalytic domain Usually, the loops that link β-strands to the adjacent helices carry amino acid residues of the active site; some of these loops may be long enough to be considered as domains in their own right Thus,

in most cases where the structure has been determined by crystallography, a large loop between the third β-strand and third helix is discussed as a separate domain, domain B This loop has an irregular structure that varies from enzyme to enzyme,

and, it has been argued, should not always be considered a separate domain but, in some cases, should be thought of as part of a structural unit containing other loops Similarities in domain B amongst members of sub-groups of the α-amylase family have, however, been found, e.g., various glucosidases resemble each other, while a relationship can be demonstrated between enzymes such as neopullulanase and cyclomaltodextrinase

Figure 2 3 The domain architecture of α-amylases

2.1.4.2 The catalytic mechanism of amylases:

Catalytic steps in glycoside bond cleavage in retaining enzymes The proton donor protonates the glycosidic oxygen and the catalytic nucleophile attacks at C1 leading to formation of the first transition state The catalytic base promotes the attack

of the incoming molecule ROH (water in hydrolysis or another sugar molecule in transglycosylation) on the formation of the covalent intermediate resulting in a second

Figure2.4 The catalytic mechanism of α-amylases

The amylase enzymes are among the most industrially important enzymes, having wide applications, such as in brewing, starch processing, textile, alcohol production, and detergent industries In the past decades, we have seen a shift from the acid hydrolysis of starch to the use of starch-converting enzymes in the production

of maltodextrin, modified starches, or glucose and fructose syrups The conditions prevailing in the industrial applications in which enzymes are used are rather extreme, especially with respect to temperature and pH Therefore, there is a continuing demand to improve the stability of the enzymes and thus meet the requirements set by specific applications Most of the well-characterized starch degrading enzymes are from thermophilic and hyperthermophilic prokaryotes and much less research have been devoted to enzymes from other extremophiles such as halophiles The structural

information from the H orenii kind of extremophiles would be very useful for us to

design highly stable and commercially useful proteins

In this thesis, we report the structure of AmyA at both low and high salt concentrations at 1.6 and 1.83 Å resolution, respectively The analysis of AmyA structure reveals a novel surface feature and its implications for stability under poly-extreme conditions We also report the biophysical characterization studies of AmyA under a

broad salinity range and this provides insight into the stability of AmyA over the entire salinity range and at high temperatures

2.2.1 Protein purification

The AmyA gene was cloned in the pTrcHisB vector (Invitrogen) by amplifying the gene by PCR using H.orenii’s genomics DNA and the protein was

over expressed with an N-terminal hexahistidine tag in Escherichia coli strain TOP10

cells (Invitrogen) One colony of TOP10 cells containing the pTrcHis-AmyA construct was used to inoculate 10 ml of LB medium with 100 µg ml-1 of ampicillin (LB-Amp medium) and the cells were grown at 37 °C for 16 h This 10 ml culture was added to 1 L of fresh LB-Amp medium and the cells were grown at 37 °C to an

OD600 of 0.6 The protein was induced for 4 h by adding thiogalactopyranoside (IPTG) to a final concentration of 1 mM The cells were harvested by centrifugation at 5000g for 10 minutes and resuspended in 20 ml phosphate buffer [20 mM sodium phosphate (pH 7.8), 500 mM NaCl] The cells were lysed at 4 °C using a French press at 6.9 MPa DNaseI was added to the lysate at a concentration of 5 µg ml-1 and the sample was incubated on ice for 30 minutes to increase the precipitation of heat denatured protein Any insoluble material was removed by centrifugation at 10,000g for 15 min The supernatant was incubated at

isopropyl-β-D-65 °C for 30 min to denature all other E coli proteins and transferred to ice for 30

minutes to maximize protein precipitation AmyA remains active at temperatures of

up to 70 °C Therefore most of the E coli host proteins were precipitated by the

heat-precipitation technique and removed by subsequent centrifugation at 10,000g for 20 minutes The clear supernatant was loaded to a column containing 2 ml Ni-NTA

agarose affinity resin (Qiagen) After washing with phosphate buffer containing 10

mM imidazole, the recombinant AmyA protein was eluted from the column with 200

mM imidazole in the phosphate buffer The protein was further purified by filtration chromatography using a Hiload 16/60 Superdex75 column (Amersham Pharmacia Biotech) with buffer consisting of 20 mM HEPES (pH 7.5), 500 mM NaCl The His-tag was not removed before crystallization and the yield of the protein was 2mg/litre of culture The pure protein fractions were pooled together and concentrated to a final concentration of 5 mg ml-1 using Centriprep and Centricon YM-10 devices (Millipore)

gel-2.2.2 Crystallization and data collection

The AmyA protein sample (at 5 mg ml-1, assayed by the Bradford method) was set up for crystallization using the Crystal Screen Cryo kit (Hampton Research) with the vapour-diffusion hanging-drop method 1 µl of protein sample was mixed with 1 µl of reservoir solution and equilibrated against 700 µl of the reservoir solution

at 24 °C Single crystals formed under the condition 70 mM sodium acetate (pH 4.6), 5.6% PEG 4000 and 30% glycerol The condition was further optimized by mixing 4

µl of the protein sample with 1 µl of the reservoir solution Crystals were obtained with maximum dimensions of 0.1 x 0.1 x 0.6 mm after 1 d (Fig 2.2) The mother liquor with glycerol increased by 5% was used as a cryoprotectant The crystals were flash-cooled in liquid nitrogen X-ray diffraction data were collected at ALS,

Berkeley at -173 °C The crystal belongs to the orthorhombic P212121 space group and contains one molecule per asymmetric unit All data were indexed, integrated and scaled using the programs DENZO and SCALEPACK (Otwinowski and Minor, 1997) The data collection and crystallographic statistics are summarized in Table 2.1

Figure 2.5 lAmyA crystal picture Crystals of AmyA, with maximum

dimensions of 0.1 x 0.1 x 0.6 mm

Table 2.1 Crystal parameters and data collection statistics of

AmyA at low salt Values in parentheses are for the last resolution

Wavelength (Å) Resolution of data (Å)

No of measured reflections

No of unique reflections Redundancy

Completeness (%) Mean I/σ(I)

99-1.6 (1.65-1.6) 153,954

65,039 2.37

96 (87.8) 10.8 (5.2) 0.062 (0.179)