Báo cáo khoa học: Interaction of the E2 and E3 components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus ppt

Perham, Department of Biochemistry, University of Cambridge, Sanger Building, Old Addenbrooke’s Site, 80 Tennis Court Road, Cambridge CB2 1GA, UK Fax: +44 1223 338707 Tel: +44 1223 33863

dehydrogenase multienzyme complex of Bacillus

stearothermophilus

Use of a truncated protein domain in NMR spectroscopy

Mark D Allen, R William Broadhurst, Robert G Solomon and Richard N Perham

Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, UK

The 2-oxo acid dehydrogenase complexes consist of

multiple copies of three distinct enzymes that together

catalyse the oxidative decarboxylation of 2-oxo acids,

in the presence of thiamine diphosphate (ThDP),

coen-zyme A (CoA), Mg2+ and NAD+, to generate CO2

and the corresponding acyl-CoA The complexes are

assembled around an oligomeric [octahedral (24-mer)

or icosahedral (60-mer)] dihydrolipoyl acyltransferase

(E2) core to which multiple copies of the relevant

2-oxo acid decarboxylase (E1) and dihydrolipoyl dehy-drogenase (E3) bind tightly, but noncovalently, to form the intact multienzyme complexes [1] The pyru-vate dehydrogenase (PDH) complex has a pivotal role

in most organisms, catalysing the irreversible reaction that links the glycolytic pathway and the tricarboxylic acid cycle Pyruvate is oxidatively decarboxylated to acetyl-CoA, which can be either broken down further

in the tricarboxylic acid cycle or used as an important

Keywords

pyruvate dehydrogenase; protein–protein

interaction; NMR spectroscopy;

multienzyme complex; protein domains

Correspondence

R.N Perham, Department of Biochemistry,

University of Cambridge, Sanger Building,

Old Addenbrooke’s Site, 80 Tennis Court

Road, Cambridge CB2 1GA, UK

Fax: +44 1223 338707

Tel: +44 1223 338635

E-mail: r.n.perham@joh.cam.ac.uk

(Received 19 July 2004, revised 27 September

2004, accepted 28 September 2004)

doi:10.1111/j.1432-1033.2004.04405.x

A 15N-labelled peripheral-subunit binding domain (PSBD) of the dihydro-lipoyl acetyltransferase (E2p) and the dimer of a solubilized interface domain (E3int) derived from the dihydrolipoyl dehydrogenase (E3) were used to investigate the basis of the interaction of E2p with E3 in the assem-bly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearo-thermophilus Thirteen of the 55 amino acids in the PSBD show significant changes in either or both of the 15N and 1H amide chemical shifts when the PSBD forms a 1 : 1 complex with E3int All of the 13 amino acids reside near the N-terminus of helix I of PSBD or in the loop region between helix II and helix III 15N backbone dynamics experiments on PSBD indicate that the structured region extends from Val129 to Ala168, with limited structure present in residues Asn126 to Arg128 The presence

of structure in the region before helix I was confirmed by a refinement of the NMR structure of uncomplexed PSBD Comparison of the crystal structure of the PSBD bound to E3 [Mande SS, Sarfaty S, Allen MD, Perham RN & Hol WGJ (1996) Structure 4, 277–286] with the solution structure of uncomplexed PSBD described here indicates that the PSBD undergoes almost no conformational change upon binding to E3 These studies exemplify and validate the novel use of a solubilized, truncated pro-tein domain in overcoming the limitations of high molecular mass on NMR spectroscopy

Abbreviations

E1, pyruvate decarboxylase; E2, dihydrolipoyl acetyltransferase; E3, dihydrolipoyl dehydrogenase; E3int, dimer of a solubilized interface domain; PDH, pyruvate dehydrogenase; PSBD, peripheral subunit-binding domain; ThDD, thrombin-cleavable di-domain; ThDP, thiamine diphosphate.

metabolite in the synthesis of fatty acids, cholesterol,

steroids in eukaryotes and N-acetyl-derived

carbo-hydrates

The structural and mechanistic core of the 2-oxo

acid dehydrogenase complexes is provided by the E2

component, each chain of which is composed of three

independent domains At the N-terminus are 1–3

tan-demly repeated lipoyl domains, followed by a

peri-pheral subunit-binding domain (PSBD) responsible for

binding E3 in the majority of organisms In

icosahe-dral complexes, the PSBD is also involved in the

bind-ing of E1 [1,2] The catalytic (acyltransferase) core

domain, which assembles to form the octahedral or

icosahedral inner core of the complexes, is proposed to

bind E1 in the octahedral complexes [2,3], and is

loca-ted at the C-terminus Each of the individual domains

is separated by long and flexible linker regions, which

make possible active site coupling by allowing for large

movements of the lipoyl domain(s) [1,4–6]

To date it has proved impossible to obtain crystals

of an intact PDH complex However, several

three-dimensional structures have been determined for the

individual domains of E2 chains from both icosahedral

and octahedral complexes: lipoyl domain structures

from Bacillus stearothermophilus, Escherichia coli and

Azotobacter vinelandii PDH complexes [7–9], and

E coli and A vinelandii 2-oxoglutarate dehydrogenase

complexes [10,11]; PSBD structures from E coli

2-oxo-glutarate dehydrogenase and B stearothermophilus

PDH complexes [12,13]; the octahedral acyltransferase

core from A vinelandii PDH [14] and E coli

2-oxo-glutarate dehydrogenase [15] complexes and the

icosa-hedral core from the B stearothermophilus PDH

complex [16] Additionally, E3 and E1 structures from

a number of sources have been solved by X-ray

crys-tallography [17–24], and it has been possible to obtain

a crystal structure of a B stearothermophilus E3–PSBD

complex formed between a lipoyl domain-PSBD

di-domain and an E3 dimer [24] Most recently,

mod-els for the overall structures of the assembled PDH

complexes, of some 10 MDa in molecular mass, have

been proposed based on cryoelectron microscopy data

[25,26]

An E3 dimer can bind only one PSBD domain of

E2 [1,27] This is due to steric hindrance, the

associ-ation with one PSBD close to the twofold axis of E3

preventing the association of a second PSBD [28,29]

The interaction occurs at the C2-axis of symmetry in

the E3 dimer, chiefly with the interface domain of E3,

which is highly conserved and generates the majority

of contacts across the dimer interface A surface loop

region of the PSBD appears to undergo a

conforma-tional change when PSBD binds to the E3 dimer, as

judged by the observed differences between the struc-tures obtained by NMR spectroscopy for the free PSBD [13] and X-ray crystallography for the E3–PSBD complex [28]

This paper describes the interaction of a15N-labelled PSBD (residues 119–171) of the B stearothermophilus E2p polypeptide chain with the dimer of a protein domain (E3int) representing the interface domain (resi-dues 343–470) of B stearothermophilus E3 The use of the engineered E3int domain (27 kDa as the dimer) was introduced because the high molecular mass (112 kDa) of the intact E3 dimer limited the applica-tion of NMR spectroscopy Chemical shift differences between the backbone resonances of the uncomplexed PSBD and PSBD bound to E3int indicate that several amino acids near the N-terminus of helix I of the PSBD are at or near the E3-binding site, confirming and extending the earlier crystal structure [28] The backbone dynamics of PSBD were also investigated, as the rigidity or otherwise of the proposed E3-binding site is crucial to a proper understanding of protein– protein interactions within the multienzyme complex Further, an improved solution structure of PSBD was subsequently determined and compared with that of the PSBD in the crystal structure of the PSBD–E3 complex [28], thereby allowing a better estimate of the extent of molecular rearrangement that accompanies binding to E3

Results and Discussion

Interaction of PSBD with the intact E3 dimer E3 (0.1 nmol of dimer) was mixed with various amounts of PSBD before being subjected to nondena-turing polyacrylamide gel electrophoresis, as described elsewhere [30] Saturation of binding, as evidenced by the appearance of free PSBD in the Coomassie-stained gel, was found to occur when 0.1 nmol of E3 dimer was mixed with 0.1 nmol of PSBD As expected, there-fore, free PSBD interacts with B stearothermophilus E3 in a 1 : 1 stoichiometry identical to that observed previously with the PSBD as part of the lipoyl-PSBD di-domain [30]

The changes in backbone amide 15N and1H chem-ical shifts upon mixing PSBD with E3 were slight (maximum chemical shift changes are 0.213 p.p.m and 0.029 p.p.m for 15N and1H shifts, respectively; results not shown) Significant chemical shift changes (greater than 0.10 and 0.02 p.p.m in the 15N and 1H dimen-sions, respectively) were observed for Asn126, Arg127, Ala131, Gly156 and Glu161 However, for all but the eight and two residues in the N- and C-terminal

regions, respectively, linewidths in the PSBD–E3 dimer

(10 : 1) mixture were much broader than those found

for PSBD alone, greatly diminishing the information

that could be derived from the spectrum This is

pre-sumably due to the high molecular mass (115 kDa)

and correspondingly long rotational correlation time

of the PSBD–E3 complex

Interaction of PSBD with E3int

To overcome these problems connected with the high

molecular mass of the PSBD–E3 complex, the

inter-action of PSBD with a solubilized interface domain

(E3int) of B stearothermophilus E3 was studied The

E3int domain comprises the C-terminal portion,

resi-dues 343–470, of the B stearothermophilus E3 chain

and, based on the crystal structure of E3, contains a

majority of the intersubunit contacts in the E3 dimer

[1,18,28] It has proved possible to generate a

dimer-ic form of the corresponding interface domain of the

homologous glutathione reductase, a flavoprotein like

E3, from E coli To achieve this, hydrophobic

pat-ches on the surface of the domain were altered to

display charged or hydrophilic residues in key

posi-tions, thereby rendering the domain soluble but not

prone to aggregation beyond the desired dimer stage

[31] A similar programme of mutation was therefore

undertaken on a subgene encoding the interface

domain of the B stearothermophilus E3 The final

version of the solubilized E3int domain contains

seven mutations of surface hydrophobic residues

(I384D, V352N, L391A, I465G, I384D, V352N and

D443N) chosen to render the domain more soluble

than its wild-type counterpart, but not to interfere

with dimerization The E3int domain forms a dimer

in the same way as E3, as determined by gel

filtra-tion (data not shown), and can be expected to

inter-act with PSBD in essentially the same way as the

intact E3 dimer [29,30] However, the E3int dimer

has a molecular mass of only 27 kDa compared with

110 kDa for E3

Linewidths for all but the 12 and terminal residues

in the N- and C-terminal regions of PSBD (which do

not form part of the structured region) were found to

be narrower than those observed on complexation

with E3 This reflects the smaller size and shorter

rotational correlation time of the PSBD–E3int

com-plex (molecular mass 32 kDa) However, linewidths

were still broader than those in the spectra of PSBD

alone

The changes in backbone amide 15N and 1H

chem-ical shifts upon mixing PSBD with E3int are shown in

Fig 1A,B, respectively The chemical shift changes

between the free PSBD and PSBD–E3int spectra are more pronounced (maximum changes are 0.500 p.p.m and 0.065 p.p.m for 15N and 1H shifts, respectively) than those observed between free PSBD and PSBD–E3 spectra Significant changes in chemical shift were observed for Ala123, Asn126, Arg127, Arg128, Val129, Ile130, Ala131, Met132, Val135, Arg136, Lys137, Lys154 and Arg157 The location of residues undergo-ing large chemical shift changes upon interaction with E3 and E3int are mapped on to the three-dimensional structure of PSBD in Fig 1C (where changes greater than 0.10 p.p.m or 0.02 p.p.m in the 15N and 1H dimensions, respectively, are indicated by grey spheres) The major locations include residues 126–137 straddling the start of helix I (residues 134–142) and residues Lys154, Gly156 and Arg157 in the loop (L2) between helix III (residues 145–149) and helix II (resi-dues 159–168) As shown in Fig 1C, all the affected residues lie relatively close in space, with both the region near the N-terminus of helix I of the domain and the three residues in loop L2 contributing to the E3-binding site

Backbone dynamics of PSBD The region (Met132 to Ser134) before the start of helix I of the PSBD structure was reported previously [13] to be largely unstructured owing to an absence of detectable long-range NOEs in the NMR spectrum The availability of uniformly 15N-labelled PSBD enabled us now to analyse the backbone dynamics using a steady-state [1H]-15N NOE experiment [32,33]

in 20 mm potassium phosphate buffer, pH 6.5, at 298

K The calculated values of g for PSBD plotted against residue number are shown in Fig 2 The plot indicates that the structured region extends from Val129 to Ala168, but residues Asn126, Arg127 and Arg128 do appear to be partly mobile

Structure determination of PSBD Because the backbone dynamics experiment revealed that residues 117–125 and 171 of the PSBD are highly flexible in solution, only residues 126–170 were inclu-ded in the structure calculations The final set of

conformational constraints (summarized in Table 1) The final ensemble comprised 25 structures, all of which had no distance violations > 0.25 A˚, no dihed-ral angle violations > 5
, and very small deviations from ideal covalent geometry Most of the residues are restricted to favoured or additionally allowed regions

of (/,w) space with, of the nonglycine residues, only

Lys154 consistently falling into disallowed regions.

Figure 3 illustrates the ensemble and secondary

struc-ture regions of PSBD

All statistical calculations were carried out on the

structured region from Val129 to Ala168 Outside this

region the torsion angles of / and w deviated rapidly away from their mean values The average root mean square (rms) deviation from the mean coordinates for backbone nuclei over the structured region from Val129 to Ala168 is 0.35 A˚ (Table 1)

-4.0

-3.6

-3.2

-2.8

-2.4

-2.0

-1.6

-1.2

-0.8

-0.4

0.0 120 125 130 135 140 145 150 155 160 165 170

Residue number

Fig 2 Backbone dynamics of PSBD Plot of the backbone steady state { 1 H}- 15 N NOE enhancement, g, for PSBD as a function of residue number.

-0.06

-0.04

-0.02

0.00

0.02

0.04

0.06

K154 R127 V129

I130

A131 M132

V135

K137

B

Residue Number

120 125 130 135 140 145 150 155 160 165 170

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

Residue Number

N126

V129

I130

R136

R157

A123

120 125 130 135 140 145 150 155 160 165 170

Lys137

Arg136 Val135

Met132 Ala131

Ile130

Val129 Arg 128 Arg127 Asn126

Lys154

Gly156

Arg157

Glu161

N

C

Fig 1 NMR spectroscopy of the interaction of PSBD and E3int Plots of the (A) 15 N and (B) 1 H N chemical shift changes between the [15N,1H]-HSQC spectra of free PSBD and the PSBD–E3int dimer (10 : 1) mixture as a function of residue number (C) Schematic MOLSCRIPT

[46] drawing of the three-dimensional structure of PSBD; residues undergoing significant chemical shift changes (> 0.10 p.p.m or

> 0.02 p.p.m in the 15 N or 1 H dimensions, respectively) are illustrated as grey spheres The molecule is coloured from the N- to the C-termi-nus following the colours of the visible spectrum (violet for N-termiC-termi-nus and red for the C-termiC-termi-nus).

Description of the three-dimensional structure

of PSBD

The refined NMR structure of PSBD is consistent with

the two previously reported E3-binding domain

struc-tures: that of synthetic peptides representing the

PSBD of the dihydrosuccinyltransferase chain of the

2-oxoglutarate dehydrogenase complex of E coli [12]

and of the dihydroacetyltransferase chain of the PDH complex of B stearothermophilus [13] The domain consists of two a-helices comprising residues Ser134 to Lys142 (helix I) and Leu159 to Leu168 (helix II), and

a short 310-helix Asp145 to Val149 (helix III), with loops connecting the helices

The N-terminal region (Val129 to Pro133) is relatively well-defined and possesses a hydrogen bond between Val158 HNand Ile130 C¢ This hydrogen bond was not observed for the construct used by Kalia et al [13] and undoubtedly the inclusion of this restraint in our struc-ture calculations permitted this region to adopt a more rigid conformation The region is further stabilized by a number of hydrophobic contacts between Val129, Ala131, Val135 and Ile146 The loop L2 between the

310-helix and helix II contains several residues with amide protons exhibiting reduced rates of exchange with the solvent Analysis of the initial structures allowed hydrogen bond acceptors for each of these residues to

be identified The structure also appears to be stabilized

by hydrogen bonds between Tyr138 OgHgand Asp164

Od2(although this was not included as a restraint in the structure calculations)

Structural comparisons The three-dimensional structure of the PSBD from the E2 chain of the B stearothermophilus PDH complex has been determined before, by NMR spectroscopy of the free PSBD [13] and X-ray crystallography of the E3–PSBD complex [28] All three structures now avail-able are very similar with respect to the arrangement

of structural motifs and loops, with the exception of loop L2 where differences exist Previously it was noted that upon superposition of the NMR structure and crystal structure, the tip of loop L2 appeared to

Table 1 Summary of constraints and statistics for the 20 accepted

structures of B stearothermphils PSBD domain.

Structural constraints

Distance constraints for 22 hydrogen bonds 44

Statistics for accepted structures

Statistics parameter (± SD)

Rms deviation for distance constraints 0.006 A ˚ ± 0.001A˚

Rms deviation for dihedral constraints 0.02
± 0.01

Mean X-PLOR energy term (kcalÆmol)1± SD)

E (dihedral and TALOS constraints) 0.003 ± 0.002

Rms deviations from the ideal geometry (± SD)

Average atomic rmsd from the mean structure (± SD)

Residues 129–168 (N, Ca, C atoms) 0.33 A ˚ ± 0.09 A˚

Residues 129–168 (all heavy atoms) 0.33 A ˚ ± 0.09 A˚

Ser134

Lys142 Asp145

Ala168

Leu159 Val149

Fig 3 Solution structure of PSBD from

NMR spectroscopy Superposition of

back-bone traces of the 25 accepted structures

over residues 129–168, and a MOLSCRIPT [46]

representation of the PSBD structure in the

same orientation The residues defining the

secondary structural elements are labeled.

move by 9.2 A˚ [28] It was suggested that the

differ-ence might be due to changes taking place in loop L2

upon binding to the E3 dimer or might reflect an

intrinsic flexibility of loop L2 The results from the

backbone dynamics experiment described above,

how-ever, reveal that the loop is inherently rigid on the

sub-nanosecond time scale Together with the absence of

significant chemical shift changes for more residues in

loop L2 when the PSBD binds to E3int, this suggests

that the loop does not undergo significant structural

rearrangement upon binding to E3 Moreover,

super-positioning shows that the crystal [28] and present

NMR (see above) structures of PSBD are virtually

identical Comparative Ramachandran plots of the

PSBD NMR and crystal structures reveal only minor

differences in loop L2, which are confined to residues

Gly153, Lys154 and Asn155

The differences observed between the previous

NMR structure [13] and that determined here (see

above) are probably due to the use of uniformly

15N-labelled PSBD and the inclusion of additional

resi-dues in the construct at the N-terminus of the domain

In particular, the absence of residues Arg127 and

Arg128 from the earlier construct [13] may have

affec-ted the stability of the N-terminal region, thereby

pre-venting it from adopting the defined conformation

observed in the crystal and new NMR structures The

different conformations of loop L2 between the two

NMR structures can also be attributed to the different

N-terminal regions, as the lack of the observed

hydro-gen bond constraint between Val158 HNand Ile130 C¢

would have significantly affected the earlier

calcula-tions [13] The remaining slight differences between the

crystal structure and the new NMR structure are

prob-ably due to the relatively low number of NOEs

observed between Lys154 and Asn155 in the loop L2

The site of interaction between E3 and PSBD

The chemical shift changes observed on formation of a

complex between PSBD and E3int are likely to be due

principally to direct contact between the two proteins,

together with some changes in the conformation of

PSBD upon binding Figure 1 shows large chemical shift

changes for Asn126, Arg127, Val129, Ile130, Ala131,

Met132, Val135, Arg136, Lys137, Lys154, Gly156 and

Arg157, all of which are located close in

three-dimen-sional space The crystal structure of the

E3-lipoyl-PSBD di-domain complex has already been determined

[28] The hydrophobic residues Val129, Ile130, Ala131,

Met132, Val135 and basic residues Arg127, Arg128,

Arg136, Lys137, Lys154 and Arg157 implicated by these

NMR studies are arranged in the crystal structure such

that the hydrophobic regions on PSBD and E3 form multiple van der Waals contacts, while the acidic resi-dues of E3 and basic resiresi-dues of PSBD generate multiple salt-bridges The results obtained by means of NMR spectroscopy are now wholly consistent with the struc-ture of the PSBD–E3 complex determined by X-ray crystallography This rules out any doubt that the crys-tal structure might not be a valid representation of the interaction in solution and indicates that the interaction between PSBD and E3, though very tight (Kd
10)9m [1,30]), is of the direct ‘lock-and-key’ kind rather than

an induced fit As a corollary, it is clear that the approach we have been developing, of creating a soluble construct containing the interface region of the E3 dimer [31,34], has made it possible to overcome the molecular mass limitation in using NMR spectroscopy to study protein structure and protein–protein interaction This augurs well for future studies, for example by transverse relaxation compensated NMR spectroscopy

Experimental procedures

Materials Bacteriological media were from Difco (Detroit, MI, USA) The pBSTNAVDD vector carrying the dihydrolipoyl ace-tyltransferase gene that encodes residues 1–171 of B stearo-thermophilus E2p was generated earlier [35] Plasmid pET11d and E coli host strain BL21(DE3) [(F–, ompT, hsdSB(rb , mb), gal, dcm (DE3)] were obtained from Nov-agen Inc (Madison, WI, USA)

Construction of expression vector pET11ThDD Construction of plasmid pET11ThDD encoding residues 1–171 of B stearothermophilus E2p with a thrombin-cleavage site (LVPRGS) in place of Ala118 proceeded via double overlapping PCR mutagenesis using standard techniques [36] Plasmid pBSTNAVDD [35] was used as the template DNA The PCR product was digested with NcoI and BamH1, purified by means of agarose gel electrophoresis and ligated into vector pET11d previously digested with NcoI and BamH1 and treated with calf intestinal alkaline phospha-tase The resulting vector encodes the sequence of a di-domain with a thrombin-cleavable linker region (ThDD) The DNA was fully sequenced to ensure its fidelity

Expression and purification of15N-labelled PSBD

E coli strain BL21(DE3) cells transformed with pET11ThDD were grown to an A600 of 1.5 in K-Mops minimal medium [37] containing 10 mm 15N-NH4Cl before being induced by the addition of isopropyl

thio-b-d-gal-actoside (1 mm final concentration) The cells were

harves-ted after 3 h of induction, resuspended in 50 mm Tris⁄ HCl

buffer, pH 7.5, containing 1 mm EDTA, 1 mm

phenyl-methanesulfonyl fluoride and 0.02% (w⁄ v) sodium azide,

and disrupted in a French press Cell debris was removed

by centrifugation and the supernatant was fractionally

pre-cipitated with ammonium sulphate The protein that was

precipitated between 35 and 80% saturation was dialysed

overnight into 50 mm potassium phosphate, pH 7.0 ThDD

was purified by consecutive cation-exchange and

anion-exchange chromatography using Hi-loadTM-S and MonoTM

-Q columns, respectively [38] Purified ThDD (10 mgÆmL)1)

was treated with thrombin, 40 UÆmL)1 final concentration,

in 20 mm potassium phosphate buffer, pH 7.0, at 37
C for

6 h, to cleave the linker between the lipoyl domain and

PSBD PSBD released in this way was purified by

cation-exchange chromatography and its purity checked by

SDS⁄ PAGE [38]

Generation of the E3int dimer

B stearothermophilus E3 was purified from an

over-expres-sion system in E coli described previously [39] The dimeric

interface domain, comprising residues 343–470 of B

stearo-thermophilus E3 [34] was prepared in essentially the same

way as the dimeric interface domain was excised from the

homologous E coli glutathione reductase and rendered

more soluble by appropriate mutations on its freshly

exposed hydrophobic surfaces [31] To achieve this for the

B stearothermophilus E3 interface domain, the following

amino acid exchanges were made to its freshly exposed

hydrophobic surfaces: four residues making hydrophobic

contacts with the FAD domain (L389D, A390S, L391A

and I465G) and three residues abutting the NAD or central

domains (I348D, V352N and I443N) The interface domain

with these seven changes was designed to retain one of the

two C2axes of symmetry present in the intact wild-type E3

dimer

NMR spectroscopy

For 2D NMR spectroscopy, samples of15N-labelled PSBD

(5 mm) and unlabelled PSBD (8 mm) in 20 mm potassium

phosphate buffer, pH 6.5 (90% H2O⁄ 10% D2O, v⁄ v) were

used Two-dimensional [1H]15N-HSQC spectra, used to

detect backbone and side-chain amide resonances which

slowly exchange with D2O, were recorded in 20 mm

potas-sium phosphate buffer, pH 6.5, in 99.996% (v⁄ v) D2O

NMR spectra were recorded on a Bruker AM-500

spectro-meter (500.13 MHz for 1H and 50.68 MHz for 15N) at

298 K Mixing times were 60 ms and 150 ms for the TOCSY

and NOESY experiments, respectively Proton and nitrogen

chemical shifts were determined relative to sodium

2,2-di-methyl-2-silapentane-5-sulfonate and liquid ammonium,

respectively Sequential assignment of the cross-peaks was

achieved using interresidue NOE connectivities by standard 2D NMR procedures [40,41] Stereospecific assignments of

Hb resonances were determined by the use of cross-peak intensities in the 2D NOESY and 2D TOCSY [40] The back-bone dynamics of PSBD were investigated using steady-state {1H}-15N nuclear Overhauser enhancement (NOE) experi-ments [32,33] Values of the {1H}-15N NOE were determined according to the formula g¼ (I ¢ – Iref)⁄ Iref, where I¢ is the intensity of a cross-peak in an experiment with 3 s broad-band1H presaturation and Irefis the intensity in a reference spectrum recorded without presaturation

NMR spectroscopic studies of protein–protein interaction

For 2D NMR spectroscopic studies of protein–protein interaction, samples of 15N-labelled PSBD (1 mm) in

20 mm potassium phosphate buffer, pH 6.5 (90%

H2O⁄ 10% D2O, v⁄ v), were used B stearothermophilus E3 dimer (final concentrations of either 0.25 mm and 0.1 mm) was added to PSBD samples to study the interaction of PSBD with E3 B stearothermophilus E3int dimer (final concentration 0.1 mm) was added to PSBD samples to study the interaction of PSBD with E3int

Distance constraints

A set of distance constraints was derived from NOESY spectra recorded in H2O and D2O with mixing times of

150 ms Each NOESY spectrum was integrated according

to the cross-peak strengths and calibrated by comparison with NOE connectivities obtained for standard interresidue distances within an a-helix After calibration, the NOE con-straints were classified into four categories: 1.8–2.8, 1.8–3.5, 1.8–4.75 and 2.5–6.0 A˚ The distance constraints in the ini-tial ensemble of structure calculations were derived only from NOESY cross-peaks that could be unambiguously assigned on the basis of chemical shift alone

Structure calculation and refinement Structure refinement proceeded in an iterative manner in which distance constraints were added or modified follow-ing analysis of the previous ensemble of structures The vic-inal proton coupling constant (3JaN) was determined by the use of a series of 2D J-modulated (15N-1H)-COSY spectra [42] Comparison of the coupling constant with an experi-mentally derived Karplus curve [43,44] enabled the torsion angle, /, to be estimated when the coupling constant was greater than 7.1 Hz

In the final round of structure calculations, hydrogen bond constraints were included for a number of backbone

HN groups whose signals were observed to change slowly when the sample buffer was exchanged for D2O For

hydrogen bond partners, two distance constraints were used

where the distance (D)H-O(A) corresponded to 1.8–2.1 A˚

and (D)N-O(A) to 2.8–3.2 A˚ The stereospecific assignments

of Hb resonances determined from NOESY and TOCSY

spectra were confirmed by analysing the initial ensemble of

structures Additional stereospecific assignments were

iden-tified for resolved resonances when the side-chain atoms

were sufficiently well-defined in the ensemble of structures

Each round of assignment was followed by a set of

struc-ture calculations with the structural constraints including

the stereospecific assignment, and confirmed when the

resulting structures did not show any distance violations

greater than 0.25 A˚ The 3D structure of PSBD was

calcu-lated from 841 experimental constraints using cns version

1.1 [45] Twenty structures were calculated from an

exten-ded conformation using torsion angle dynamics in a

standard simulated annealing protocol Stereospecific

assignments of prochiral protons were used, where

avail-able; otherwise r)6 averaging was used over all equivalent

protons Structures were accepted where no distance

vio-lation was greater than 0.25 A˚ and no dihedral angle

viola-tions > 5
The final coordinates have been deposited in

the Protein Data Bank (PDB accession no 1w3d)

Acknowledgements

We thank the Biotechnology and Biological Sciences

Research Council (BBSRC) for the award of a research

grant (to RNP) and a Research Studentship (to MDA)

The core facilities of the Cambridge Centre for

Molecu-lar Recognition were funded by the BBSRC and The

Wellcome Trust We are grateful to Mr C Fuller for

skilled technical assistance and to Dr ARC Raine for

his help with the structure calculation

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