Fluorescence studies show that human activation domain interacts with the FMN-binding domain of human methionine synthase reductase hMSR.. Key differences in the sequences and structures
Trang 1activation domain of human methionine synthase
Kirsten R Wolthers1,*, Helen S Toogood1,*, Thomas A Jowitt1, Ker R Marshall2, David Leys1and Nigel S Scrutton1
1 Faculty of Life Sciences, University of Manchester, UK
2 Department of Biochemistry, University of Leicester, UK
Human methionine synthase (EC 2.1.1.13;
5-methyl-tetrahydrofolate homocysteine methyltransferase, hMS)
plays a vital role in folate metabolism and the
recyc-ling of homocysteine It is the only enzyme that
liberates tetrahydrofolate (H4folate) from
methyltetra-hydrofolate (CH3-H4folate), which is a key metabolite
for protein and nucleic acid biosynthesis The enzyme
contains a cobalamin cofactor, and in the highly nucle-ophilic cob(I)alamin state, the cofactor abstracts a methyl group from CH3-H4folate to form H4folate and methylcob(III)alamin (Scheme 1, 1a) [1,2] The methyl group is subsequently transferred from methylcob(III)alamin to homocysteine to generate methionine and cob(I)alamin (Scheme 1, 1b) [3]
Keywords
activation domain; cobalamin-dependent
enzyme; methionine synthase; methionine
synthase reductase; S-adenosyl-methionine
Correspondence
N S Scrutton, Manchester Interdisciplinary
Biocentre and Faculty of Life Sciences,
University of Manchester, 131 Princess
Street, Manchester M1 7ND, UK
Fax: +44 161 306 8918
Tel: +44 161 306 5152
E-mail: nigel.scrutton@manchester.ac.uk
Database
The atomic coordinates and structure
fac-tors (202K) have been deposited in the
Pro-tein Data Bank, Research Collaboratory for
Structural Bioinformatics, Rutgers
Univer-sity, New Brunswick, NJ, USA (http://
www.rcsb.org)
*These authors contributed equally to this
work
(Received 3 October 2006, revised 22
November 2006, accepted 28 November
2006)
doi:10.1111/j.1742-4658.2006.05618.x
Human methionine synthase (hMS) is a multidomain cobalamin-dependent enzyme that catalyses the conversion of homocysteine to methionine by methyl group transfer We report here the 1.6 A˚ crystal structure of the C-terminal activation domain of hMS The structure is C-shaped with the core comprising mixed a and b regions, dominated by a twisted antiparallel
b sheet with a b-meander region These features, including the positions of the active-site residues, are similar to the activation domain of Escheri-chia coli cobalamin-dependent MS (MetH) Structural and solution studies suggest a small proportion of hMS activation domain exists in a dimeric form, which contrasts with the monomeric form of the E coli homologue Fluorescence studies show that human activation domain interacts with the FMN-binding domain of human methionine synthase reductase (hMSR) This interaction is enhanced in the presence of S-adenosyl-methionine Binding of the D963E⁄ K1071N mutant activation domain to the FMN domain of MSR is weaker than with wild-type activation domain This suggests that one or both of the residues D963 and K1071 are important in partner binding Key differences in the sequences and structures of hMS and MetH activation domains are recognized and include a major reorien-tation of an extended 310-containing loop in the human protein This struc-tural alteration might reflect differences in their respective reactivation complexes and⁄ or potential for dimer formation The reported structure is
a component of the multidomain hMS : MSR complex, and represents an important step in understanding the impact of clinical mutations and poly-morphisms in this key electron transfer complex
Abbreviations
AdoMet, S-adenosyl-methionine; AUC, analytical ultracentrifugation; FLD, flavodoxin; FNR, ferredoxin-NADP+oxidoreductase; hMS, human methionine synthase; MALLS, multiangle laser light scattering; MS, methionine synthase; MSR, methionine synthase reductase.
Trang 2The latter half of the reaction highlights additional
roles of MS in cell homeostasis (a) the production of
methionine, which is an essential amino acid and a
precursor in the biosynthesis of S-adenosyl-methionine
(AdoMet); and (b) the recycling of homocysteine,
which is cytotoxic to vascular endothelial cells and an
independent risk factor in coronary arterial disease
[4,5] High total plasma homocysteine coupled with
diminished folate pools has also been associated with
an increased incidence of neural tube defects in
new-borns and Down’s syndrome [6,7]
These medical conditions may arise from either a
vitamin deficiency or inborn errors in the gene
enco-ding hMS or the gene encoenco-ding the enzyme involved in
the reactivation of hMS, human methionine synthase
reductase (hMSR) [8] The activity of hMS ceases after
1–2000 catalytic turnovers with the one-electron
oxi-dation of cob(I)alamin (Scheme 1, 2) [3,9] Human
MSR binds to hMS forming a ‘reactivation complex’
and an NADPH-derived electron is shuttled to
cob(II)alamin via the FAD and FMN cofactors of
MSR (Scheme 1, 3) [9] Transfer of a methyl group
from AdoMet accompanies reduction by MSR, thus
converting cob(II)alamin to methylcob(III)alamin and
returning MS to the primary catalytic cycle
Reactiva-tion of the Escherichia coli homologue of hMS, MetH,
also involves FAD and FMN redox centres; however,
the cofactors are components of individual proteins:
ferredoxin-NADP+ oxidoreductase (FNR) and
flavo-doxin (FLD), respectively [10]
To date, the majority of information on the
struc-tural and functional behaviour of hMS has been
derived from biochemical and biophysical research on
MetH [11] Although the 3D structure of the
full-length MetH has not been determined, structures of
three individual functional modules have been solved
The N-terminal module, determined from MetH
of Thermotoga maritima, consists of two b,a barrels,
which each house substrate-binding pockets for homo-cysteine and CH3-H4folate [12] The cobalamin is sandwiched between two domains in the central mod-ule [13] Finally, the C-terminal domain, termed the activation domain, binds AdoMet and forms part of the reactivation complex with hMSR [14] The struc-ture of the E coli MetH activation domain is C-shaped, with a twisted antiparallel b sheet as a cen-tral feature AdoMet binds near the centre of the inner surface of the domain and is held in place by interac-tions with both side-chain and backbone atoms [14] MetH, and by extension MS, are envisioned to be highly dynamic proteins as both substrate-binding pockets on the N-terminal module (separated by 50 A˚) and the activation domain have to form discrete com-plexes with the cobalamin-binding domain in order to catalyse each of the transmethylation reactions of the primary catalytic cycle and reactivation process [15,16] hMS has an added level of complexity compared with MetH, as its redox partner, MSR is itself a multi-domain protein; MSR is modelled on the structural family of diflavin reductases, of which cytochrome P450 is the prototype [17] Enzymes belonging to this class of proteins have a NADPH⁄ FAD-binding domain, tethered to an FMN-binding protein, which are related to bacterial FNR and FLD, respectively Studies have shown that with the two-component system of E coli, FLD forms mutually exclusive com-plexes with MetH and FNR [18] If the FMN-binding region of MSR behaves similarly, this domain would pivot between hMS and the FAD domain of MSR
to facilitate electron transfer Alternatively, the FMN domain is relatively immobile and is sandwiched between the FAD domain and hMS during electron transfer to cob(II)alamin In this case, the binding interfaces between the activation domain of hMS and MSR may be sufficiently different from that of the MetH activation domain and FLD
Evidence for notable differences in the binding inter-face between MetH and hMS is supported by the poor ability of the FNR⁄ FLD-reducing system to reactivate hMS and the complete inability of MSR to reactivate MetH [19] Structural information on the hMS activa-tion domain will help establish those key components
on these proteins that make them specific for their respective redox partners In addition, structural infor-mation will help identify how particular clinical and polymorphic (e.g P1173L) variations appearing in the
MS activation domain compromise the activity of the enzyme and lead to various clinical states [20,21] Here, we provide insight into the biophysical proper-ties of human MS⁄ MSR system and we report the crystal structure of a D963E⁄ K1071N double-mutant
CH3-H4-folate
H4-folate
Cob(I)alamin
Methylcob(III)alamin
Methionine
Homocysteine Cob(II)alamin
e
AdoMet AdoHyc
e
1a
2
3
1b
Scheme 1.
Trang 3of the 38 kDa activation domain of hMS to 1.6 A˚
resolution Solution studies demonstrate binding of
both the wild-type and mutant activation domains to
the FMN domain of the physiological partner protein,
MSR We show that the human activation domain
exists as a distribution of monomeric and dimeric
forms, with the monomer comprising the main
compo-nent in solution Key structural differences between
the hMS activation domain and the E coli homologue
are discussed
Results
Crystal structure determination
The mutant activation domain structure of hMS was
determined by molecular replacement using the
struc-ture of the corresponding domain of MetH as a model
[14] The asymmetric unit contains two protein
mole-cules, related by near-perfect translational symmetry
The crystallographic and final refinement statistics are
summarized in Table 1 The last 1–2 residues are not
visible in the electron-density map Similarly, residues
T1001–G1003 and D1072–A1074, which are located in
flexible loops, are not visible In humans, residue
Gln1041 in both subunits is in a disallowed region in
the Ramachandran plot It is one of only two residues
in the sharp turn between b2 and b3 in the b meander
In several organisms, including E coli, this residue is glycine (G1008) which can accommodate this geometry The structure of the activation domain monomer of human MS is C-shaped, the central feature comprising
a twisted antiparallel b sheet (Fig 1A), similar to the
E coli structure [14] This is not surprising as the two
MS proteins share 48% sequence identity in the activa-tion domain, with the human activaactiva-tion domain con-taining several insertions (Fig 1B) The core of the structure comprises mixed a + b regions, dominated by
an antiparallel b sheet, with an overall topology that does not resemble any other AdoMet-binding protein structure [14] An antiparallel b sheet (b1, b2, b5 and b8) forms the upper part of the structure along with strands b3 and b4, which form a b meander On the opposite side of the sheets, after the meander, is a region of six a helices and a 310-helix Helices a2–a4 connect the strands b1 and b2, whereas helices a5–a7 follow strand b5 The long helix a6 is surrounded by the central b sheet on one side, b6–b7 and the short helix a7
on the other side The C-terminus is dominated by two short helices a10–a11 [14] The two mutations D963E and K1071N are located after b1 and b4, respectively,
in regions distant from the AdoMet-binding site Owing
to their location in flexible surface loops, these muta-tions are not thought to have a major impact on the overall structure, but rather have only localized effects
Structural comparison of the hMS and E coli MetH activation domains
Figure 2A shows a superimposition of the structures
of the hMS activation domain and corresponding MetH domain in E coli Using the program dalilite [22], the rmsd value of the superimposition is 2 A˚ with
a Z-score of 41.9 (314 residues aligned by Ca) Although the two structures look very similar, there are significant differences between them, most strik-ingly in the region of helices a3–a4 In the human enzyme, helix a3 is extended by a further four amino acids followed by the insertion of an extra five amino acids between a3 and the 3102-helix This has resulted
in a dramatic reorientation of these latter two helices, relative to the E coli enzyme, beginning at residue Leu984 (Leu956 in MetH) and ending at the beginning
of the a4 helix In the E coli enzyme, this region is oriented so the 310-helix is within 4 A˚ of helix a7, and within 7 A˚ of Ile1126 in the AdoMet-binding region
In the human structure, the longer a3 helix ends at the beginning of the extra five amino acids The 310 2-helix is oriented below the beginning of 2-helix a4 and is
Table 1 Crystallographic data and refinement values for hMS
activation domain double-mutant D963E ⁄ K1071N All numbers in
parentheses represent last outer shell (1.66–1.60 A ˚ ).
D963E ⁄ K1071N mutant Data collection
Resolution limits (A ˚ ) 20–1.6 (1.66–1.60)
R merge (%) 6.4 (40.2)
Refinement
Rfac⁄ R free (%) 20.9 ⁄ 22.3 (27.0 ⁄ 29.9)
rmsd bond lengths (A ˚ ) 0.006
Average B-factor (A˚2 ) 25.2
Ramachandran plot
Most favoured regions (%) 94.1
Additionally allowed regions (%) 5.6
Generously allowed regions (%) 0.0
Disallowed regions (%) 0.3 (Q1041 both subunits)
Trang 4close to the three amino acid turn between helices a5–
a6 (Fig 2A) This positions the 310-helix at one of the
tips of the C-shaped molecule, preventing it from being
in a position near the AdoMet-binding region as in
MetH This creates a new hydrophobic cluster not
found in the E coli enzyme between residues Phe993
and Phe997 and the a3 and a6 helices Other
hydro-phobic residues involved include Trp982, Val1009,
Tyr1121, and Ile1124 Part of this region is disordered
in the human enzyme, with clear electron density
lack-ing for residues Thr1001–Gly1003
The role of this loop)310region in E coli is
uncer-tain, but owing to its location near the
AdoMet-bind-ing site it may be involved in interactAdoMet-bind-ing with its FLD
partner Recent cross-linking and NMR studies of
MetH show that Lys959 (Lys987 in hMS) is located
close to Glu61 of FLD [18,23] in the activation
domain–FLD complex, supporting the notion that this
is a key interaction in the formation of the reactivation
complex hMS interacts with a much larger partner,
MSR, which is a cytochrome P450 reductase-like
pro-tein constructed by the fusion of two genes encoding a
FMN-containing FLD and an FAD-containing ferre-doxin oxidoreductase separated by a large interdomain linker [9,17] The change in position of this loop)310 region of the human enzyme in addition to the shift in position of Lys987 from the equivalent MetH residue Lys959 of 7.9 A˚ (Fig 2A) suggests a different mode of interaction between human MS and MSR compared with the E coli complex Because no structure is avail-able for MSR, or any structure of a MS–partner com-plex, the exact nature of these interactions remains to
be determined
The region containing b3–b5, known as the b mean-der [14], contains a six amino acid insertion in the human structure (Fig 1A) The b-meander begins with
a twist in b2 at the conserved residue Pro1036 (Pro1003
in E coli), as indicated by an asterisk in Figs 1B and 2A This meander is oriented 90 to the central sheet [14] In the human enzyme, three of these extra residues are located between b3 and b4, forming an additional solvent-exposed 310-helix (3103), absent in the E coli enzyme The other three extra amino acids are located
in the more disordered region between b4–b5, next to
A
B
Fig 1 (A) Stereoview of the overall
struc-ture of the hMS activation domain The
car-toon is drawn as a gradient from blue at the
N-terminus to red at the C-terminus Red
spheres, location of the mutations D963E
and K1071N; magenta sphere, clinical
muta-tion P1173L (B) Sequence alignment of
human and E coli MS activation domains,
confirmed by structural superimposition.
Secondary structure elements were
assigned using DSSP [28] Pink, secondary
structure in humans; green, major
differ-ences in secondary structure in E coli
compared with humans; orange, additional
3 10 -helices in humans; asterisk, Pro1036
(beginning of b-meander); blue, residues
interacting with AdoMet in E coli.
Trang 5the mutation K1071N The equivalent lysine residue in
E coli activation domain (Lys1035) is suggested to be
involved in the interaction between MS and FLD [18]
We note that in the two regions of the protein where
the major differences occur between the E coli and
human structures, both contain a residue which in
MetH is known to interact with FLD This suggests
that these differences between both structures of the
activation domains might reflect structural differences
in their respective reactivation complexes
Like MetH, the structure of the hMS activation
domain contains some well-ordered water molecules in
two cavities which interact with residues in the
b-mean-der region This region, containing helices a10–a11, is
one of the more conserved regions of the activation
domain It contains many buried ionic and polar
resi-dues, forming an extended network of salt bridges and
hydrogen bonds similar to the E coli enzyme [14]
Active-site region
A superimposition of the two structures at the MetH
AdoMet-binding site shows a high similarity of both
residue type and position (Fig 2B) AdoMet binds near the centre of the inner surface of the domain
of the E coli structure (Fig 2A) It is held in place
by hydrophobic interactions and hydrogen bonds, through both side chain and backbone interactions, and is partly solvent exposed [14] This is one of the more conserved regions of the activation domain and contains the consensus sequence Arg-X-X-X-Gly-Tyr critical for the binding of AdoMet [14] The human enzyme structure does not contain AdoMet, although the positions of the active site residues are strikingly similar
Only two of the residues (Tyr1190 and Ala1141) known to directly interact with AdoMet in the E coli protein are different in human activation domain (i.e Phe1228 and Ser1179, human numbering) However, these residues in the E coli protein interact with Ado-Met via backbone interactions only The main struc-tural difference in the active site residues is the reorienting of the Tyr1177 side chain away from where AdoMet would bind This suggests only minimal chan-ges in the positions of the active site residues are required for binding of AdoMet
A
B
Fig 2 Stereoview of a superimposition of human and E coli MS activation domains (A) Overall structures: blue and green car-toons are human and E coli proteins, respectively; AdoMet from the E coli struc-ture (1MSK) [14]; is shown as red sticks; Ca atoms of K987 and K959 of human and
E coli activation domains, respectively, are shown as red spheres (B) AdoMet-binding region: E coli residues interacting with Ado-Met, and the equivalent human residues, are shown as atom-coloured sticks with green and orange carbons, respectively; AdoMet is shown as purple lines.
Trang 6Monomer interactions in the crystalline state
The crystal structure reveals that both monomers
within the asymmetric unit are involved in close
inter-actions with symmetry-related molecules, leading to a
putative dimeric form Figure 3A shows two views of
the structure of this human MS activation domain
‘dimer’ The contact surface between the two
mono-mers, consisting of two nearly identical interaction
sites, is 609 A˚3 with a shape complementarity (Sc)
value of 0.769, which is in the range of Sc values for
surfaces within known dimeric proteins and
protein–lig-and interactions [24] There were no other interactions
of sufficient size between monomers of the activation
on the structure, and their symmetry-related molecules
to be able to accurately calculate Sc values Both the
size of the contact surface, the Sc value and the fact
both monomers in the asymmetric unit form near
iden-tical dimers suggests dimer formation could be
physio-logically relevant rather than simply a consequence of
the crystallization conditions and⁄ or crystal packing
The central cavity of the dimer consists of a large
elliptical-shaped central groove, with extensions at the
base of the dimer forming a cross shape This
exten-sion of the cavity is due to the side chains of several
residues of the extended a3 helix of both subunits
pointing directly into the cavity The volume of this cavity is calculated to be 7000 A˚3using the program CASTp [25] The smallest part of the cavity (24· 5 A˚)
is at the lower region of the dimer (Fig 3A) close to where AdoMet binds in the E coli structure [14] Two AdoMet molecules can be modelled to bind within the dimer, with the large size of the cavity easily allowing substrate entry However, the side chains of residues Tyr988 and Lys987 of the second molecule of the
‘dimer’ clash with the region where the adenosyl group
of AdoMet is bound in the MetH structure While the side chain of Lys987 can move to accommodate Ado-Met, the side chain of Tyr988 cannot, suggesting the dimeric form is not compatible with substrate binding Superimposition of the E coli structure onto the human activation domain ‘dimer’ shows significant cla-shes due to the reorientation of the a3–310loop region (Fig 3A) Clearly, the E coli activation domain is unli-kely to form a human-like dimer Figure 3B shows the residues involved in forming the dimer interface of the human activation domain Although there is a signifi-cant contact surface between the monomers, the number
of direct interactions is small The key interacting resi-due is Arg991 The NH2 group of Arg991 forms a hydrogen bond with the carbonyl oxygen of Glu1077 (2.7 A˚) The NH1 and NH2 groups are also within van
A
B
Fig 3 Dimeric nature of human MS
activa-tion domain (A) Two views of the human
activation domain dimer, with the
mono-mers shown as green and blue cartoons.
AdoMet is superimposed onto the structure
as red sticks in the equivalent position
found in the E coli enzyme [11] E coli
‘dimer’ superimposed on the human dimer
is represented by an olive cartoon (B) The
dimer interface of human activation domain
shown as green and blue cartoons for
monomers A and B, respectively Side
chains of residues directly interacting at the
dimer interface are shown as atom-coloured
sticks Side chains of residues lining a
hydrophobic pocket are shown as magenta
sticks.
Trang 7der Waal’s distance of the hydroxyl group of Tyr1079,
as is the NE group to the backbone oxygen atoms of
Pro1078 and Glu1077 The OD2 atom of Asp1120
forms a salt bridge with the NH2 group of Arg927
(2.7 A˚) The interface is furthermore formed by
hydro-phobic interaction between Tyr988 of one subunit with
Pro1173, Tyr1177, Pro1178 and Tyr1227 of the second
subunit As the equivalent residues of the E coli
activa-tion domain are located in significantly different
posi-tions, these dimer interface interactions are not present
Molecular mass determination and dimer
formation
A key question to address is whether the dimer species
as seen is a significant species in solution To answer
this question, we carried out multiangle laser light
scattering (MALLS) and analytical ultracentrifugation
(AUC) to determine the proportion of dimer formed
in solution under both high and low salt conditions
Table 2 shows the results of the sedimentation and
light-scattering analyses Both wild-type and the
dou-ble-mutant activation domains show near identical
sedimentation profiles (Fig 4A) with corrected
sedi-mentation coefficients (s0
20;W) of 2.87 ± 0.16 and 2.84 ± 0.2, respectively, in 0.4 m sodium acetate
buf-fer (i.e similar to the crystallization conditions)
How-ever, in NaCl⁄ Pi and Tris⁄ HCl buffers in the absence
of sodium acetate, the sedimentation coefficient was
increased to 2.98 ± 0.2 Thus, the molecular mass of
the two proteins are identical, as shown by light
scat-tering, and there is no detectable difference in shape
between the wild-type and mutant proteins in the same
buffer systems (Fig 4B) However, differences in the
solution properties are seen for both proteins in
differ-ent buffers The addition of 0.4 m sodium acetate to
the buffers increases the presence of the dimer species
(14% of the total protein analysed) The estimated
frictional ratios for the monomeric species using the
sedimentation coefficient distribution (c(s)), where
f⁄ f0¼ estimated frictional ratio, for 2D size and shape distributions showed that there is a difference in the apparent length or flexibility of the molecules in the two different buffers In 0.4 m sodium acetate, the molecules exhibited a more extended or more flexible conformation with a frictional ratio of 1.45 compared with a value of 1.31 representing a more compact structure in NaCl⁄ Pi This corresponds to a difference
in hydrodynamic radius of 3 A˚ (Table 2)
Global analysis of mutant and wild-type protein in high salt buffer at different concentrations was per-formed to ascertain whether there was evidence of reversible association occurring between monomeric and dimeric species The results fitted well to a mono-mer–dimer model However, only a very small amount
of dimer is present This was true even at very high concentrations of 2 mgÆmL)1, giving a very low associ-ation constant This indicates that, although there may
be dimer species present within the samples in 0.4 m sodium acetate, there is no real evidence for a dynamic associating system
The molecular mass obtained throughout these experiments was lower than expected (Table 2) for the sequence molecular masses when taking into account the calculated mass for hydrated protein This might be attributed to a small amount of protease cleavage during purification The molar mass distribution is near identi-cal for the wild-type and mutant proteins giving an aver-age monomeric molecular mass of 41 600 ± 1020 Da The high polydispersity detected is similar to the results gained using sedimentation equilibrium indicating that a proportion of the molecules are slightly smaller than sequence molecular mass (Fig 4C) Some of the dimer species can also be seen using this technique
Analysis of dimer interactions by chemical cross-linking
Inspection of the dimer interface reveals that Lys925
of one subunit of the activation domain ‘dimer’ is
Table 2 Solution studies of wild-type and mutant activation domain to determine oligomeric states in high- and low-salt buffers NaCl ⁄ P i ; M, observed hydrated mass of the monomer; S, sedimentation coefficient; R H , radius of the structure; f ⁄ f o , estimated frictional ratios; ND, not determined; NA, not applicable.
R
Experimental
M (kDa)a M (kDa)b S020,W R Ha(nm) R Hb(nm) f ⁄ f 0
Bead modelling Monomer
Dimer
R H (nm) S020,W
a Determined by sedimentation studies b Determined by light scattering c 100 m M Tris ⁄ HCl pH 7.0 + 400 m M Na acetate.
Trang 8likely to be in close proximity to the N-terminal amino
group of the second subunit To determine if this
spe-cific surface interaction occurs frequently in solution,
an attempt was made to cross-link these two specific primary amine groups with a homo-bifunctional imi-doester cross-linker Mass analysis of in-gel trypsinized samples showed that although the 80-kDa band observed in SDS–PAGE was indeed a dimer of activa-tion domain molecules, the locaactiva-tion of the cross-link indicated a ‘back-to-back’ orientation for the two monomers and is thus inconsistent with the crystal structure (results not shown) This suggests that either: (1) the concentration of the dimer species under cross-linking conditions is very small; (2) the location of the N-terminus in solution is not consistent with models of the dimer (the N-terminus is not visible in the crystal structure of the activation domain); and⁄ or (3) there is some proteolytic removal of the N-terminus during purification
Analysis of the interaction between MS activation domain and the FMN domain of MSR Titration of the activation domain of MS (both the D963E⁄ K1071N mutant and wild-type proteins) with the FMN domain of human MSR resulted in a quenching of the fluorescence emission spectra of the flavin cofactor The decrease in fluorescence intensity showed a hyperbolic dependence on the concentration
of the activation domain (Fig 5) A fit of Eqn (1) to the data yielded the apparent dissociation constant for the complex formed between activation domain and the FMN-binding region of MSR (Table 3) The wild-type activation domain has an apparent Kd value 1.3 lm for the FMN domain, which is 10-fold lower than the apparent Kd (11.9 lm) for binding of the D963E⁄ K1071N to the flavin-binding protein The presence of AdoMet in the fluorescence bind-ing assays resulted in a twofold decrease in the apparent dissociation constant for both the wild-type (0.7 lm) and the mutant (4.7 lm) activation domains
Discussion
hMS is important for maintaining adequate levels of methionine and AdoMet, preventing the accumulation
of cytotoxic homocysteine, and is essential in methi-onine metabolism Elevated levels of homocysteine in the blood have been linked to an increased likelihood
of developing cardiovascular disease, birth defects, Down’s syndrome and affecting the development of some types of cancer [4–7,26] Functional deficiency
of MS or MSR results in diseases such as homocys-tinuria, hyperhomocysteinemia and hypomethionine-mia [8,20] A P1173L mutation (magenta sphere in
A
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Sedimentation coefficient (s)
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Sedimentation coefficient (S)
0 50000 100 000 150 000 200 000
0.0
2.0x10 -5
4.0x10-5
6.0x10 -5
8.0x10-5
1.0x10 -4
1.2x10-4
Molar Mass (Da)
B
0
20 000
40 000
60 000
80 000
10 0000
Volume (ml)
0.0 0.2 0.4 0.6 0.8 1.0
C
Fig 4 (A) Sedimentation coefficient distribution c(s) of wild-type
(solid line) and mutant (dotted line) in 10 m M Tris, 150 m M NaCl
pH 7.0 Inset shows the same protein run in 0.4 M sodium acetate.
(B) Wild-type molar mass distribution c(M) using the estimated
fric-tional ratio (f ⁄ f 0 ) of 1.45 (C) Wild-type (solid line) and mutant
(dot-ted line) elution from a Superdex 200 gel filtration column in
NaCl⁄ P i +0.4 M sodium acetate, with the absolute molar mass
superimposed The thick dotted line within the major peak shows
the degree of polydispersion of the enzyme in NaCl ⁄ P i
Trang 9Fig 1B) in the activation domain of human MS is
commonly found among patients exhibiting
hype-rhomocysteinemia [21] This residue is located
between Arg1172 and Ala1174, both of which interact directly with AdoMet in the E coli structure (Fig 2B) P1173L is located at the start of a loop in the active site and which contains four proline resi-dues of the sequence P-X-P-X-X-P-X-X-P This sequence is highly conserved among MS enzymes sug-gesting an evolutionary pressure to retain this struc-ture in the active site of the activation domain Other known clinical mutations of the human activation domain include H920D, E1204X (early termination), and insertion⁄ deletion mutations [21]
The suggestion of a dimer of human activation domain in the crystal structure as well as in some solu-tion studies raises some important issues As the acti-vation domain is the C-terminal domain of MS, separated from the rest of the protein by a 38-residue linker region, a key question to be addressed is the possibility of the full-length enzyme forming a dimeric structure Also, the possibility of binding Ado-Met ± the FMN domain of MSR influencing the like-lihood of dimer formation needs to be considered That the major differences between the hMS and MetH activation domains occur in those regions involved in dimer association is intriguing This might reflect the need to recognize different redox partners, i.e FLD versus MSR, but a role in dictating the oligo-meric state of MS cannot be ruled out That said, only
a small proportion of dimer is found in solution stud-ies, and this is most prevalent under high salt condi-tions Clearly, further structural analysis of other components of the MS holoenzyme is required to ascertain if full-length hMS possesses higher order quaternary structure
Fluorescence titration assays have demonstrated an interaction between the FMN-binding domain of MSR and the activation domain of MS The appar-ent Kd value determined from these binding assays is similar to that reported for the interaction between
E coli FLD and its redox partner, MetH [23] The decrease in dissociation constant for the complex with the wild-type and mutant form of the activation domain in the presence of AdoMet suggests that binding of the substrate for the transmethylation reaction might effect a conformational change in the activation domain to increase its affinity for the FMN domain of MSR
The 10-fold higher dissociation constant measured for the mutant activation domain⁄ FMN domain inter-action indicates that Asp963 and⁄ or Lys1071 are important residues in this interaction Several lysine mutants of E coli FLD have also showed a marked decrease (3- to 70-fold) in affinity towards MetH [23], suggesting that salt bridges are a key recognition
0
2
4
6
8
10
A
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
B
Fig 5 Fluorescence titration of the FMN domain of MSR with the
wild-type (A) and D963E ⁄ K1071N double-mutant (B) of the MS
acti-vation domain The FMN domain at 0.25 l M was titrated with both
forms of the purified activation domain under conditions described
in the Experimental procedures The binding assays were
per-formed in the absence (d) or presence (s) of 1 m M S-adenosyl
methionine The change in the FMN fluorescence intensity was
plotted versus the concentration of the activation domain and the
curves show the best fit of the data to the quadratic Eqn (1).
Table 3 lists the calculated dissociation constants for the wild-type
and D963E ⁄ K1071N alone and in the presence of AdoMet.
Table 3 Fluorescence titration of the FMN domain of MSR with
the wild-type and D963E ⁄ K1071N double-mutant of the MS
activa-tion domain The FMN domain at 0.25 l M was titrated with both
forms of the purified activation domain under conditions described
in the Experimental Procedures.
Enzyme
K d (l M )
No AdoMet
1 m M AdoMet
Trang 10feature at the binding interface in both the E coli and
human systems
While this study describes the structure of a
double-mutant of the activation domain, the location of the
mutations are on flexible, surface-exposed loops We
conclude from this observation, combined with the
MALLS and AUC data which showed that the
wild-type and mutant activation domains have identical
shapes, that the structure of the mutant domain
resem-bles closely that of the wild-type activation domain
To further understand the process of MS
reactiva-tion, a structure of the cob(I)alamin ± activation
domains in complex with at least the FMN domain of
MSR is needed (studies that we are currently pursuing)
The structure of the activation domain reported here
has allowed us to gain insight into the likely mode of
AdoMet binding, and provide atomic level insight into
the effect of clinical mutations on the activity of MS
Experimental procedures
Cloning and mutagenesis
The cDNA encoding the activation domain of methionine
synthase gene was cloned by PCR amplification, using
nondegenerate oligonucleotides based on the published
sequence Total RNA was isolated and purified from whole
human blood using a High Pure RNA isolation kit (Roche
Diagnostics, Welwyn Garden City, UK) cDNA was
gener-ated by reverse transcription using the Titan RT PCR
sys-tem (Roche Diagnostics) The MS gene was cloned into the
plasmid vector pET15b, and the 1184 bp PCR product
cor-responding to the 3¢-terminus of the MS gene was
gener-ated with Pfu turbo DNA polymerase (Stratagene, La
Jolla, CA) and ligated into a pGEMT vector (Promega,
Madison, WI) to generate the vector pGEMMB The
sequence encoding the activation domain (residues 925–
1265) was subsequently amplified by PCR from the
pGEMMB vector, incorporating the restriction sites NcoI
and HindIII into the 5¢- and 3¢-regions, respectively These
restriction sites were used to subclone the PCR product
into pET23d, generating clones containing a C-terminal
His-tag Sequencing of the resulting vector, pACT, revealed
two missense mutations that resulted in conversion of an
Asp at position 963 to a Glu and a Lys to an Arg at
posi-tion at 1071 (At this stage we cannot say if the identified
differences to the published sequence of human methionine
synthase (GenBank accession number Q99707) represent
polymorphic variation of the MS gene.) The pACT vector
was subsequently mutated to revert the sequence back to
wild-type Both the wild-type gene and the uncorrected
into competent E coli BL21(DE3) (Stratagene) MSR
FMN domain was cloned as a glutathione-S-transferase
fusion protein according to the method of Wolthers et al [27]
Protein production and purification
Wild-type and mutant activation domain proteins were
ampi-cillin Cells were lysed by sonication in buffer A (20 mm
inhibitor tablets (Roche) and Benzonase (Merck Bio-sciences Ltd., Nottingham, UK) The proteins were puri-fied by running through Ni-NTA resin contained in 5 mL HisTrap column (Amersham Biosciences, GE Healthcare, Little Chalfont, UK) in Buffer A Proteins were eluted in
a gradient from 0 to 0.5 m imidazole For the final puri-fication step, the protein was loaded onto HiPrep Q Sepharose (Amersham Biosciences, GE Healthcare) in
gradi-ent from 0 to 1 m NaCl
The expression and purification of the FMN domain of MSR was carried out according to a modification of the method of Wolthers et al [27] An additional purification step was performed using Resource Q (Amersham Bio-sciences, GE Healthcare) in Buffer B The protein was
elut-ed in a gradient of 0–0.5 m NaCl
Crystallogenesis and data collection
Crystals of the wild-type and mutant MS activation domains were grown using the sitting drop vapour diffusion
into 10 mm Tris pH 7.0 containing 0.1 mm EDTA and 0.5 mm dithiothreitol The reservoir solution comprised 0.1
10–12% poly(ethylene glycol) 8000 and 10–12% poly(ethy-lene glycol) 1000 Crystals appeared between 2 and 14 days Crystals were soaked in mother liquor supplemented with 5% poly(ethylene glycol) 200 as a cryorotectant, before being flash-cooled in liquid nitrogen A full 1.6 A˚ data set was collected on a single crystal of the mutant MS activa-tion domain at the European Synchrotron Radiaactiva-tion Facil-ity (Grenoble, France) on ID14-EH1 using an ADSC Q4 CCD detector Owing to problems associated with twin-ning, a model could not be obtained for data collected from wild-type crystals
Structure determination and refinement
Data were processed and scaled using the HKL package programs denzo and scalepack [28] The structure was solved via molecular replacement using the program
activa-tion domain from E coli MetH [14] as the search model