Tài liệu Báo cáo khoa học: Crystal structures of the regulatory subunit of Thr-sensitive aspartate kinase fromThermus thermophilus pdf

Abbreviations AK, aspartate kinase; BsAKII, aspartate kinase II from Bacillus subtilis; CgAK, aspartate kinase from Corynebacterium glutamicum; CgAKb, regulatory subunit of aspartate kin

Thr-sensitive aspartate kinase from Thermus thermophilus Ayako Yoshida1, Takeo Tomita1, Hidetoshi Kono2, Shinya Fushinobu3, Tomohisa Kuzuyama1and Makoto Nishiyama1,4

1 Biotechnology Research Center, The University of Tokyo, Japan

2 Computational Biology Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, Kyoto, Japan

3 Department of Biotechnology, The University of Tokyo, Japan

4 RIKEN SPring-8 Center, Hyogo, Japan

Aspartate kinase (AK; EC 2.7.2.4) is an enzyme that

catalyzes the first committed step, the phosphorylation

of the c-carboxyl group of aspartate, of the biosynthetic

pathway of the aspartic acid group amino acids Lys,

Thr, Ile, and Met, in microorganisms and plants AK

is classified into two groups according to subunit

orga-nization: homo-oligomer or heterotetramer AK from

Thermus thermophilus (TtAK), AK from C

glutami-cum(CgAK) and AKII from Bacillus subtilis (BsAKII)

are heterotetramers containing equimolar amounts of

a-subunits and b-subunits [1–3], whereas AKIII from Escherichia coli (EcAKIII), AKI from Arabidopsis thaliana and AK from Methanococcus jannaschii (MjAK) are homo-oligomers of identical subunits [4–6] AK of the a2b2 type is encoded by in-frame overlapping genes, so that the amino acid sequence of the b-subunit is identical to about 160 amino acids of the C-terminus of the a-subunit As seen in other enzymes involved in the first step in amino acid bio-synthesis, AK is regulated through feedback inhibition

Keywords

ACT domain; allosteric regulation; crystal

structure; thermostability; threonine

biosynthesis

Correspondence

M Nishiyama, Biotechnology Research

Center, The University of Tokyo, 1-1-1

Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Fax: +81 3 5841 8030

Tel: +81 3 5841 3074

E-mail: umanis@mail.ecc.u-tokyo.ac.jp

(Received 3 November 2008, revised 10

March 2009, Accepted 31 March 2009)

doi:10.1111/j.1742-4658.2009.07030.x

Crystal structures of the regulatory subunit of Thr-sensitive aspartate kinase (AK; EC 2.7.2.4) from Thermus thermophilus (TtAKb) were deter-mined at 2.15 A˚ in the Thr-bound form (TtAKb-Thr) and at 2.98 A˚ in the Thr-free form (TtAKb-free) Although both forms are crystallized as dimers, the contact surface area of the dimer interface in TtAKb-free (3200 A˚2) is smaller than that of TtAKb-Thr (3890 A˚2) Sedimentation equilibrium analyzed by ultracentrifugation revealed that TtAKb is present

in equilibrium between a monomer and dimer, and that Thr binding shifts the equilibrium to dimer formation In the absence of Thr, an outward shift of b-strands near the Thr-binding site (site 1) and a concomitant loss

of the electron density of the loop region between b3 and b4 near the Thr-binding site are observed The mechanism of regulation by Thr is discussed

on the basis of the crystal structures TtAKb has higher thermostability than the regulatory subunit of Corynebacterium glutamicum AK, with a dif-ference in denaturation temperature (Tm) of 40C Comparison of the crystal structures of TtAKb and the regulatory subunit of C glutamicum

AK showed that the well-packed hydrophobic core and high Pro content

in loops contribute to the high thermostability of TtAKb

Abbreviations

AK, aspartate kinase; BsAKII, aspartate kinase II from Bacillus subtilis; CgAK, aspartate kinase from Corynebacterium glutamicum; CgAKb, regulatory subunit of aspartate kinase from Corynebacterium glutamicum; DSC, differential scanning calorimetry; EcAKIII, aspartate kinase III from Escherichia coli; MAD, multiwavelength anomalous diffraction; MjAK, aspartate kinase from Methanococcus jannaschii; TtAK, aspartate kinase from Thermus thermophilus; TtAKb, regulatory subunit of aspartate kinase from Thermus thermophilus; TtAKb-free, Thr-free regulatory subunit of aspartate kinase from Thermus thermophilus; TtAKb-Thr, Thr-bound regulatory subunit of aspartate kinase from Thermus thermophilus.

by end-products CgAK is regulated through concerted

inhibition by Lys and Thr [7], whereas TtAK, which is

involved in the biosynthesis of Thr and Met but

not of Lys, because T thermophilus synthesizes Lys

through a-aminoadipate as an intermediate [8,9], is

Thr-sensitive [1] In a2b2-type AK, the N-terminal

regions of the a-subunits serves as catalytic domains,

and the C-terminal regions of the a-subunits and the

b-subunits act as regulatory domains [10,11]

The regulatory domains of AK contain conserved

motifs named ACT domains that are found among

many allosteric enzymes involved in amino acid and

purine biosynthesis [12,13] The motif has a babbab

fold, and serves as a small molecule-binding domain

for allosteric regulation Several crystal structures

have been determined for enzymes containing ACT

domains; however, the mode of association between

ACT domains is quite different among the enzymes,

as summarized by Grant [14] For example, the

archetypical ACT domain association with two

side-by-side domains, each from a different chain, is

found in 3-phosphoglycerate dehydrogenase [15] In

threonine deaminase, two ACT domains in a single

peptide are arranged side-by-side to form an Ile⁄

Val-binding unit [16,17] For the ACT domain in AK,

two types of association modes are seen, as reviewed

by Curien et al [18]: one is found in

homo-oligo-meric AKs [4,6,19] and the other in a2b2-type CgAK

[20] In these ACT domains of AK proteins, there

are common structural features: (a) two ACT

domains are arranged in the C-terminal portion of a

single polypeptide; (b) the ACT1 domain is inserted

into the ACT2 domain; and (c) two ACT domains,

each from a different chain, interact to form an

effec-tor-binding unit The effeceffec-tor-binding unit of the

ACT domain in the b-subunit of CgAK (CgAKb) is

organized differently from those of homo-oligomeric

AKs In CgAKb, ACT1 and ACT2 from different

chains associate side-by-side to form an

eight-stranded b-sheet, and two eight-eight-stranded b-sheets face

each other perpendicularly In CgAKb, both

eight-stranded b-sheets are involved in effector binding On

the other hand, in homo-oligomeric AKs, two ACT1

domains from different chains associate with each

other to form an eight-stranded b-sheet, and two

ACT2 domains from different chains form an

addi-tional eight-stranded b-sheet, although these two

eight-stranded b-sheets are also arranged

perpendicu-larly and face-to-face, as in CgAKb In

homo-oligomeric AKs, only one of the eight-stranded

b-sheets is involved in effector binding Determination

of the crystal structure of the regulatory subunit of

TtAK (TtAKb) would provide information not only

on the catalytic mechanism but also on the structural features common to a2b2-type AKs

As T thermophilus is an extremely thermophilic bac-terium, proteins produced by T thermophilus have high thermostability Previously, we found that chime-ric AK, named BTT, which is composed of a catalytic domain from BsAKII and regulatory domains (a regulatory domain in the a-subunit, and a b-subunit with the same sequence as the regulatory domain) from TtAK, improved thermostability as much as wild-type TtAK [11] This result indicated that the regulatory domain of TtAK contributes not only to catalytic regulation, but also the thermostability of TtAK Comparison of the crystal structures of TtAKb and CgAKb was expected to elucidate the mechanism

of the elevated thermostability of TtAKb

In this article, we describe the crystal structures of TtAKb in two forms, Thr-bound and Thr-free, and discuss the regulatory mechanism of Thr and the struc-tural features responsible for the high thermostability

of TtAKb

Results and Discussion

Model quality The crystal structure of the Thr-bound form of TtAKb (TtAKb-Thr) was determined at 2.15 A˚ resolution, using multiwavelength anomalous diffraction (MAD) phases derived from selenomethionine (SeMet)-substi-tuted TtAKb TtAKb-Thr is a dimer containing two Thr molecules (Fig 1A), acetate molecules, which are derived from the crystallization buffer, and 153 water molecules in an asymmetric unit The electron densities

of the N-terminal (residues 1–4 in chains A and B) and C-terminal (residues 158–161 in chains A and B) sections of the structure are not seen on the map, probably owing to disorder of these regions The over-all geometry of the model according to the procheck program [21] is of good quality, with 95.4% of the res-idues in the most favored regions and 4.6% in allowed regions of the Ramachandran plot

The crystal structure of the Thr-free form of TtAKb (TtAKb-free) was determined at 2.98 A˚ resolution by molecular replacement using the structure of TtAKb-Thr as a search model The TtAKb-free crystal contains three dimers (Fig 2A), each composed of AB,

CD and EF chains, and 79 water molecules in an asymmetric unit The electron densities of the N-termi-nal (residues 1–3 in chain A, residues 1–4 in chains B and C, and residues 1–5 in chains E–G) and C-termi-nal (residues 158–161 in chains A–F, and residues 159–161 in chain B) portions of the structure are not

seen on the map The electron densities of the sections

(residues 54–56 in chains A and D, residues 53–59 in

chain B, residues 55–56 in chain C, residues 56–57 in

chain E, and residues 53–56 in chain F) of the loop

between b3 and b4 are not observed in every chain

The overall geometry of the model is good, with

89.0% of the residues in the most favored regions,

10.0% in allowed regions, 0.7% in generously allowed

regions and 0.3% in disallowed regions from

pro-check.Table 1 summarizes the refinement statistics

Overall structure

The crystal structure of TtAKb-Thr was determined as

a homodimer (Fig 1A) As TtAK is a heterotetramer

with an a2b2 configuration, where the b-subunit is

identical to the C-terminal portion of the a-subunit, as

in CgAK, the dimeric structure revealed in this study

represents the structure of the regulatory region of an

ab-heterodimer The rmsd is 0.50 A˚ between two

monomers in the asymmetric unit Structural

differ-ences between monomers are found in regions 84–88, 94–95, and 102–104 (Fig 1B) A single chain of TtAKb contains two ACT domains, ACT1 (N-termi-nal domain) and ACT2 (C-termi(N-termi-nal domain) domains The ACT domain organization of TtAKb-Thr is similar to that of CgAKb [20] but not to those of homo-oligomeric AKs ACT1 and an ACT2, each from different chains, are arranged side-by-side to form an effector (Thr)-binding unit, an eight-stranded antiparallel b-sheet with four a-helices on one side We assume that this characteristic dimer organization of the regulatory domain of AK is a feature limited to

a2b2-type AKs, because, in homo-oligomeric AKs, two equivalent ACT domains from different chains are jux-taposed to form a structural unit, and two structural units, each composed of two equivalent ACT domains, are not equivalent to each other Owing to the difference in the ACT domain arrangement, TtAKb binds two Thr molecules per dimer at two sites (site 1), each in an equivalent effector-binding unit (Fig 1A), whereas in homo-oligomeric AKs, a single

Fig 1 Overall structure of TtAKb-Thr (A) Overall structure of TtAKb-Thr The A chain and the B chain are shown in purple and green, respectively Thr molecules are shown as an orange stick model Both ACT domains forming an effector-binding unit of the front of the dimer are indicated Site 1 and site 2 of the effector-binding unit on the front are indicated by solid and dotted cir-cles (B) Superposition of two monomers in

a dimer of TtAKb-Thr ACT domains are shown by dotted circles Regions showing structural differences between monomers are indicated by solid circles.

Fig 2 Overall structure of TtAKb-free (A) Three TtAKb-free dimers in the asymmetric unit A chain, magenta; B chain, yellow; C chain, cyan; D chain, green; E chain, brown;

F chain, blue (B) EF chain dimer.

effector-binding unit binds two effectors The other

possible effector-binding sites (site 2) in the structural

unit are vacant in the TtAKb-Thr structure

The structure of TtAKb-free is also determined to

be a homodimer (Fig 2), although TtAKb was eluted

in volumes close to that of a molecular mass of

mono-mer in the absence of Thr in gel filtration

chromatog-raphy, as described below The TtAKb-free crystal

contains three dimers in the asymmetric unit, and rmsd

values for Ca among the three dimers are 0.58 A˚

between AB and CD dimers, 0.63 A˚ between CD and

EF dimers, and 0.58 A˚ between AB and EF dimers

Moreover, the rmsd values of Ca between the

mono-mers in the dimono-mers are 0.55 A˚ between the A and B

chains, 0.96 A˚ between the C and D chains, and

0.51 A˚ between the E and F chains The main

differ-ence between TtAKb-free and TtAKb-Thr is that the

residues in the loop region between b3 and b4 near the Thr-binding site are disordered in TtAKb-free, as described later

Thr-binding site

In TtAKb-Thr, the electron density of one Thr mole-cule is observed at site 1 between ACT1 and ACT2, each from different chains (Fig 3A,B) The structure

of TtAKb-Thr is quite similar to that of Thr-bound CgAKb (rmsd value of Ca is 1.73 A˚) Bound Thr mol-ecules are stabilized by ionic bonds (Asp26-Od2 for the amino group), hydrogen bonds (Gln50-Oe1 and Ile126*-O for the side chain hydroxyl group; Asn125*-Od1 and Ile126*-O for the amino group; Ile30-N, Ile126-N and Asn125*-Od1 for the carboxyl group; asterisks denote residues from another chain), and

Table 1 Data collection and refinement statistics.

Data collection

Resolution (A ˚ ) a 2.40 (2.49–2.40) 2.40 (2.49–2.40) 2.40 (2.49–2.40) 2.15 (2.19–2.15) 2.98 (3.09–2.98) Reflections (total ⁄ unique) 364 163 ⁄ 19 507 365 251 ⁄ 19 539 364 474 ⁄ 19 539 578 149 ⁄ 27 145 133 032 ⁄ 22 878

Phasing

Refinement

Average B-factor

rmsd values

Ramachandran plot e

a Values in parentheses are data of the highest-resolution shell b R sym ¼ RjI i  <I>j=R<I> c Figure of merit (FOM) was calculated with the

SOLVE program d R - factor ¼ R hkl j j F o j  F j j c j=R hkl j F o j e Calculated using PROCHECK

hydrophobic interactions (Ile24, Ile30 and Met62 for

the side chain methyl group) Two water molecules

present near the two oxygen atoms contribute to a

hydrogen bond network between the carboxyl group

of Thr, Gly29-N, Ala31-N, Ala32-N, and Phe116-O

from another chain Most of the residues and water

molecules recognizing the bound Thr in TtAKb are

conserved in CgAKb As seen in CgAKb, the carboxyl

group of Thr is located near the N-terminal section of

helix a1, suggesting that the positive charge of the

N-terminal helix dipole facilitates recognition of the

carboxyl group Importantly, Thr is bound between

two chains and is not exposed to the solvent,

suggest-ing that bound Thr plays an important role in stabiliz-ing the dimeric structure, as in CgAKb

Monomer–dimer equilibrium

In CgAKb, Thr binding induces the dimerization of CgAKb [20] Thr is bound at an effector-binding unit formed between two chains in TtAKb in a manner almost identical to that in CgAKb, suggesting that Thr binding plays a role in stabilizing the dimeric form of TtAKb To examine the effect of Thr on dimerization,

we analyzed the oligomeric state of TtAKb in the pres-ence or abspres-ence of Thr, using two different methods:

Fig 3 Thr-binding site (A) 2F o )F c map of bound Thr molecule and two water molecules The contour level of the map is 1.0r (B) Thr-bind-ing site in Thr Residues in purple are in the A chain, and residues in green are in the B chain (C) Vacant Thr-bindThr-bind-ing site in TtAKb-free Residues in blue are in the E chain, and residues in orange are in the F chain (D) Structure-based sequence alignment of TtAKb and CgAKb Alignment was performed with CLUSTALW [42], and alignment with secondary structures of TtAKb and CgAKb was performed with

ESPRIPT [43] Regions for ACT1 and ACT2 are shown by solid and broken divergent arrows, respectively.

sedimentation equilibrium by analytical

ultracentri-fugation, and gel filtration chromatography

In gel filtration, TtAKb was eluted in a volume

corresponding to a molecular mass of 21.7 kDa in the

absence of Thr (Fig 4A) The molecular mass,

esti-mated by gel filtration, is a little larger than the

calcu-lated mass of a monomer (17.7 kDa) When gel

filtration was performed in the presence of 5 mm Thr,

elution profiles gave an estimated molecular mass of

30.9 kDa

To further confirm that Thr affects monomer–dimer

equilibrium even in TtAKb, we also analyzed the

sub-unit arrangement by sedimentation equilibrium The

data fitted well with the monomer–dimer equilibrium,

with equilibrium constants of 5.6· 10)3m)1(goodness

of fit = 4.0· 10)4) and 9.1· 10)4m)1 (goodness of

fit = 2.3· 10)4) in the presence and absence of 5 mm

Thr, respectively According to the constants, TtAKb

at 1 mgÆmL)1 is mostly (91%) present as a monomer

in the absence of Thr, whereas at the same protein

concentration, 31% of TtAKb is present in a dimeric

form in solution containing Thr At 5 mgÆmL)1which

is the protein concentration used for crystallization,

58% and 27% are present as dimers in the presence and absence of Thr, respectively Thus, TtAKb is in monomer–dimer equilibrium, which is displaced by Thr and⁄ or protein concentrations; therefore, dimer formation in the crystal structure in the absence of Thr can be explained by the high concentration of TtAKb-free under crystallization conditions

CgAK is easily dissociated into a-subunits and b-sub-units during purification without Thr On the other hand, TtAK is purified in the a2b2 form even without Thr This observation suggests that binding affinity between a-subunits and b-subunits is stronger in TtAK than in CgAK; however, even TtAK showed sharp and broad elution profiles in gel filtration in the pres-ence and abspres-ence of Thr, respectively SDS⁄ PAGE of the fractions in gel filtration showed that a-subunits and b-subunits are eluted in the same volumes from the column in the presence of Thr, whereas b-subunits are eluted from the column later than a-subunits in the absence of Thr (Fig 4B–E) From these results, we conclude that b-subunits can interact with the regula-tory domains of a-subunits even without Thr, but the interaction is tighter in the presence of Thr

Fig 4 Stabilization of oligomer formation of

TtAK and TtAKb by Thr (A) Elution profiles

of TtAKb in the presence and absence of

5 m M Thr The solid line with circles and the

dotted line with squares indicate profiles in

the presence and absence of 5 m M Thr,

respectively Elution volumes for BSA

(67 kDa), chymotrypsinogen A (43 kDa),

ovalbumin (25 kDa) and ribonuclease A

(13 kDa) are indicated by a, b, c, and d,

respectively (B, C) SDS ⁄ PAGE of each

frac-tion by gel filtrafrac-tion for TtAK in the presence

(B) and absence (C) of 5 m M Thr Elution

profiles of TtAK were quantitated by IMAGEJ

[44] (D) Densitometric calibration of TtAK

subunits of SDS ⁄ PAGE in (B) The

a-sub-units and b-suba-sub-units are indicated by a solid

line with circles and a dotted line with

squares, respectively (E) Densitometric

cali-bration of TtAK subunits of SDS ⁄ PAGE in

(C) The a-subunits and b-subunits are

indi-cated by a solid line with circles and a

dotted line with squares, respectively.

Conformational change of TtAKb upon Thr

binding and its implications

Unexpectedly, the structural difference between

TtAKb-Thr and TtAKb-free is not so large: the rmsd

for Ca between two structures is about 1.5 A˚ This

contrasts with Lys-sensitive EcAKIII, which shows a

larger conformational change of the regulatory domain

dimer upon Lys binding, resulting in the displacement

of several residues responsible for catalytic function in

the catalytic domain The most distinct difference

between the structures is that the electron density of

the b3–b4 loop around the Thr-binding site is missing

in TtAKb-free, and that b-strands surrounding the

Thr-binding site show outward shifts in the absence of Thr, with 12 rotation of the ACT2 domain from the fixed ACT1 domain (Figs 3B,C and 5D) The regula-tory domain dimer of CgAK inhibited in a concerted manner by both Lys and Thr binds two Thr molecules

at site 1, like TtAKb-Thr, and easily dissociates into monomers in the absence of Thr [20] Therefore, a sim-ilar conformational change is expected for these two enzymes, depending on the presence or absence of Thr

In CgAK, mutations of the residues in the b3–b4 loop close to site 1 induced resistance to Lys or a Lys ana-log, S-2-aminoethyl-l-cysteine [20] As Lys is bound to

a vacant binding site (site 2) in the effector-binding unit composed of the ACT1 and ACT2

Fig 5 Comparison of a single effector-binding unit between TtAKb-Thr and TtAKb-free (A) Superposition of the effector-binding units of TtAKb-Thr and TtAKb-free ACT1 displays the Ca models of the B chain (residues 15–93) from TtAKb-Thr and the F chain (residues 15–93) from TtAKb-free, and ACT2 shows Ca models of the A chain (residues 5–14 and 94–157) from TtAKb-Thr and the E chain (residues 6–14 and 94–157) from TtAKb-free TtAKb-Thr and TtAKb-free are in blue and red, respectively The Thr molecule is shown as a stick model The loop between b4 and a2 corresponding to the latch loop in EcAKIII is shown as a dotted oval (B) Movement of Ca atoms caused by Thr binding mapped on the effector-binding unit of TtAKb-Thr Cyan, < 1 A ˚ ; green, < 2 A˚; yellow, < 3 A˚; orange, < 4 A˚; red, > 4 A˚ Regions showing larger movement are marked as A–E The loop between b4 and a2 corresponding to the latch loop in EcAKIII is shown as a dotted oval (C) Ca distance between TtAKb-Thr and TtAKb-free Blue indicates the distance between the A chain from TtAKb-Thr and the E chain from TtAKb-free, and red indicates the distance between the B chain from TtAKb-Thr and the F chain from TtAKb-free The regions shown

in A–E are: A, 42–45; B, 46–50; C, 51–58; D, 102–110; E, 131–134 (D) Domain motion in TtAKb caused by Thr binding The structures of TtAKb-Thr and TtAKb-free are shown in blue and pink Domain motion was analyzed by DYNDOM [45] A broken line indicates hinge axis for movement.

domains (Yoshida et al., unpublished result), the direct

function of the loop in catalytic control is unexpected

in CgAK In TtAKb, accompanied by an outward

shift of b-strands, especially b2–b4 from ACT1 near

site 1, a significant shift is also found around site 2

(Fig 5A–C) This result may suggest that two Thr

molecules bound at site 1 induce a conformational

change in site 2, thereby facilitating the binding of

additional Thr molecules at site 2 In addition, TtAKb

has a Pro-Gly sequence in the N-terminal section

(positions 28 and 29) of helix a1, which contribute to

the recognition of the carboxyl group of bound Thr by

a putative helix dipole (Fig 3B) Interestingly, the

dihedral angle of Gly29 changes upon Thr binding (for

example, / = 89.85, w =)13.64 in the A chain of

TtAKb-Thr and / = 103.16, w =)11.53 in the E

chain of TtAKb-free) In CgAKb, which can bind the

Thr molecule at site 1, the Pro-Gly sequence is

con-served at the same position (positions 27 and 28)

(Fig 3D) In addition to Pro27-Gly28, CgAKb has a

similar Pro-Gly sequence at positions 109–110 in the

N-terminal portion of helix a3 forming site 2

(Fig 3D) We also found a similar change in the

dihe-dral angle of Gly110 upon binding of Lys at site 2 of

CgAKb (details will be published elsewhere) These

observations suggest that the Pro-Gly motif functions

as a hinge to facilitate conformational change upon

effector binding in a2b2-type AKs In TtAKb, on the

other hand, the corresponding section (positions 109

and 110) has a Pro-Glu sequence (Fig 3D) Although

both dihedral angles shown by Gly29 in the presence

and absence of Thr are within the permissible range,

an allowed or generously allowed region on the

Rama-chandran plot, for Glu in general, such a marked

change upon effector binding would not be expected

for Glu At present, we cannot judge whether the

sec-ond Thr is bound to site 2 for catalytic control of

TtAK In order to further clarify the regulatory

mech-anism of TtAK by Thr, the crystal structure of

full-length TtAK in the a2b2 form is obviously required

It should be noted that, on comparison of the amino

acid sequences of CgAK and TtAK, CgAK had an

extra 11 residues at the C-terminus, forming b9,

con-sisting of a b-sheet with a b1-strand at the N-terminus

(Fig 5D) As TtAK, which is only inhibited by Thr,

does not have this extra b-strand, the b-strand may be

involved in a process of concerted inhibition by Thr

and Lys in CgAK

Comparison with other AKs

Recently, the crystal structures of Thr-sensitive MjAK

have been determined in three forms [22]: (a) complex

with magnesium adenosine 5¢-(b,c-imido)triphosphate and Asp; (b) complex with Asp; and (c) complex with Thr MjAK has a homotetrameric structure, and shows high overall structural similarity to the inhibitory complex of EcAKIII bound to Lys Although EcAKIII binds the effector, Lys, at the binding unit formed between ACT1 domains from different chains, MjAK binds Thr at the binding sites formed between ACT2 domains from different chains

In EcAKIII, transition from the R-state to the T-state, accompanied by rotational rearrangements to form a tetramer, occurs through large movement of a latch loop from the regulatory domains [4] In MjAK, however, the loop corresponding to the latch of EcAKIII is shortened, and shows no conformational change upon Thr binding Instead, Thr binding rotates the regulatory domain away from the kinase domain Accompanied by the rotation of the regula-tory domain, other loops from the catalytic domains are displaced to orient the residues important for cofactor and Asp binding in unfavorable positions Thus, in spite of their structural similarity, the regulatory mechanism is different between these homo-oligomeric AKs In TtAKb, the loop, b4–a2, corresponding to the latch in EcAKIII, is short and shows no structural rearrangement upon Thr binding (Fig 5A,B), similar to Thr-sensitive MjAK In MjAK, Thr binding causes the entire regulatory domain to rotate 6.5 away from the fixed kinase domain, resulting in opening of the catalytic site In this case, the entire domain moves as a rigid body with no significant change in the interaction between ACT domains [22] In contrast, Thr binding causes the ACT2 domain to rotate by 12 from the fixed ACT1 domain (Fig 5D), which is presumed to be the motion that closes the active site of TtAK Thus, the direction of domain motion is different between TtAK and MjAK, suggesting a different inhibitory mecha-nism in TtAK

Isothermal titration calorimetry suggests that MjAK has not only Thr-binding sites with high affinity in the regulatory domain, but also five weak Thr-binding sites per dimer, which may include a Thr bound near the Asp-binding site and those bound on the protein surface nonspecifically [22] As we have not yet determined the crystal structure of TtAK in the a2b2 form, we do not know whether TtAK also contains weak Thr-binding sites However, it should be noted that TtAK has a Ki value of less than 10 lm for Thr, which is markedly lower than that of MjAK (0.3 mm) [19] The low Kivalue of TtAK may indicate that AK activity is controlled through the high-affinity Thr site present in the regulatory domain in TtAK

Potential factors involved in the high

thermostability of TtAKb

In our previous study, we found that chimeric AK,

BTT, which is composed of the catalytic domain of

BsAKII and the regulatory domain and b-subunit of

TtAK, had thermostability as high as that of wild-type

TtAK, suggesting that the regulatory domain of TtAK

is also responsible for the thermal stability of TtAK

[11]

TtAKb and CgAKb show 36% sequence identity,

and Thr-bound crystal structures of these proteins are

very similar To understand the mechanism of

enhanced stability of TtAKb, we examined the

dena-turation of TtAKb and CgAKb by differential

scan-ning calorimetry (DSC) in the presence and absence,

respectively, of their inhibitors TtAKb has a

denatur-ation temperature approximately 40C higher than

that of CgAKb (Table 2) Both CgAKb and TtAKb

are more stable at 4.3–4.4C in the presence of Thr

Considering that TtAKb and, putatively, CgAKb are

in equilibrium between monomers and dimers, and

bound Thr shifts the equilibrium towards dimer

forma-tion, this observation indicates that the small increase

in stability results from a shift of the equilibrium to

dimer formation caused by Thr Similar protein

stabil-ization via oligomer formation has been shown for a

thermostable homoisocitrate dehydrogenase [23] In

the crystal, the contact surface area in the dimer

inter-face is larger in TtAKb-Thr (3890 A˚2) than in

TtAKb-free (3200 A˚2), indicating that Thr binding tightens the

interaction of the two chains

Many factors are involved in protein stability, such

as hydrophobic interactions [24], hydrogen and ionic

bonds [25], cavity volume [26], and other entropic

fac-tors [27] Proteins are generally stabilized by a

combi-nation of these factors [28] Among them, an increased

number of hydrogen bonds (ionic interactions) and

better internal packing are reported to be the most

important protein-stabilizing factors [29,30] To

under-stand the difference in thermostability between CgAKb

and TtAKb, we compared the crystal structures of the

two proteins When the numbers of ionic bonds and

hydrogen bonds are compared, unexpectedly, both

numbers are larger in CgAKb than in TtAKb

(Table3) In contrast, when cavity volumes were

calcu-lated from the crystal structure of TtAKb and CgAKb, the volume of TtAKb was smaller than that of CgAKb (Table 4), suggesting that TtAKb is more tightly packed than CgAKb With regard to the amino acid composition, TtAKb has a higher ratio of hydrophobic residues than CgAKb It is also remarkable that TtAKb contains more proline residues than CgAKb (Table5) Considering that most Pro residues are located at the N-termini or C-termini of loops in TtAKb (Fig 6A), the flexibility of the loop conforma-tion of TtAKb is likely to be suppressed in the dena-tured state We therefore suggest that smaller loss of entropy upon folding contributes to the stabilization

of TtAKb

We next calculated changes in Gibbs free energy from the native to the denatured state, which were esti-mated on the basis of the solvent-accessible surface area (Table 4) The difference in changes in Gibbs free energy between TtAKb and CgAKb was 22 kcalÆ mol)1, indicating that TtAKb is more stable than CgAKb A more detailed examination showed that the difference in the solvent-accessible surface area per hydrophobic amino acid residue between the native and denatured states was significantly larger in TtAKb-Thr than in Thr-bound CgAKb, whereas that

of hydrophilic residues did not change, suggesting a contribution of internal hydrophobic residues to the stability of TtAKb In fact, hydrophobicity inside the molecule was apparently higher in TtAKb-Thr than in CgAKb (Fig 6B) From these results, we conclude that better internal packing, ensured by tight

Table 2 Denaturation temperatures of TtAKb and CgAKb.

T m (C)

Table 3 Thermostabilization factors Numbers of hydrogen bonds and ionic bonds Data in parentheses are number of bonds between subunits.

Table 4 Thermostabilization factors Accessible surface area and cavity Data in parentheses are values per amino acid residue.

Difference in monomer ASA value (D )N) Hydrophobic (A˚2 ) 21 313 (70) 22 291 (68) )978 (2) Hydrophilic (A˚2 ) 6389 (21) 6709 (21) )320 (0)

Cavity volume (probe 1.4 A ˚ ) (A˚ 3 )

hydrophobic interactions in the interior of protein, and

the richness of Pro residues mainly contribute to the

stabilization of TtAKb This information might be

use-ful for the generation of more thermostable CgAK variants for industrial use

Experimental procedures

Enzyme production and crystallization

Gene cloning and the production, purification and

described [31] TtAKb-free was crystallized by the hanging drop, vapor diffusion method Crystals appeared in 0.1 m sodium acetate (pH 5.0) and 1.2–2.0 m NaCl

Data collection

The collection of TtAKb-Thr data and MAD data collection for SeMet-substituted TtAKb-Thr has been previously reported [20,31] Before data collection for TtAKb-free, a crystal was soaked briefly in cryoprotec-tant solution of 25% (v⁄ v) glycerol in reservoir solution, flash-cooled in a nitrogen gas stream at 95 K, and stored

in liquid nitrogen Diffraction data were collected with a CCD camera on the beamline NW12 of the Photon Factory AR [High Energy Accelerator Research Organi-zation (KEK), Tsukuba, Japan] Data on TtAKb-free crystals were recorded at 2.98 A˚ resolution Diffraction data were indexed, integrated and scaled using the hkl2000 program suite [32]

Structure determination and refinement

The structure of TtAKb-Thr was determined by the MAD phasing method The detailed structure determination and refinement of TtAKb-Thr were as previously described [20] TtAKb-free crystals have three dimers per asymmetric unit, and belong to the space group P31, with unit cell parame-ters of a = b = 107.2 A˚, c = 87.22 A˚, a = b = 90, and

c = 120 The structure determination for TtAKb-free by molecular replacement was performed by molrep [33] in the ccp4 program suite [34], using the model of TtAKb-Thr Subsequent refinement was conducted using the program cns1.1 [35], and model correction in the electron density map was carried out with the xtalview program suite [36] Figures were prepared using xfit in the xtal-viewprogram suite and pymol [DeLano WL, The PyMOL Molecular Graphics System (2002) at http://www.pymol org] The atomic coordinates and structure factors deter-mined in this study have been deposited in the Protein Data Bank (accession numbers 2dt9 and 2zho)

Determination of quaternary structure

The subunit organization of TtAKb was analyzed by analyt-ical ultracentrifugation and gel filtration chromatography

Fig 6 Factors important for thermostabilization of TtAKb (A) Pro

residues in TtAKb monomer (B) Pro residues in CgAKb monomer.

(C, D) Cross-sectional views of TtAKb-Thr dimer and Thr-bound

CgAKb dimer, respectively, drawn by UCSF CHIMERA [46] The surface

of the molecules is shown in cyan, and inner hydrophobic residues

are shown in pink.

Table 5 Thermostabilization factors Comparison of the amino acid

composition of TtAKb and CgAKb.