Plastic adaptation toward mutations in proteins: Structural comparison of thymidylate synthases

Perry, Kathy M.; Fauman, Eric B.; Finer-Moore, J.; Montfort, W.R.; Maley, Gladys F.; Maley, Frank; Stroud, Robert M.

doi:10.1002/prot.340080406

Cited by 161 publications

(194 citation statements)

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“…It has been previously proposed that the r.m.s. differences between the coordinates correlate with the local crystallographic B values (Chambers & Stroud, 1979;Guss, Harrowell, Murata, Norris & Freeman, 1986;Perry et al, 1990). Generally, there is a close correlation between the accuracy of atomic positions and their B values (Figs.…”

Section: S Daopin D R Davies M P Schlunegger and M G Gristtermentioning

confidence: 87%

“…The errors obtained this way often tend to be underestimated because the structures from different refinement rounds are not independent determinations. The third and very useful way is to estimate errors using a kind of error function (Stout & Jensen, 1989), such as the Luzzati plot (Luzzati, 1952), the trA plot (Read, 1986), Cruickshank's equations (Cruickshank, 1967) and the empirical B-factor equations of Perry & Stroud (Perry et al, 1990). The errors calculated from these error functions are limited to random errors that follow a normal statistical distribution.…”

Section: Accuracy Assessmentsmentioning

confidence: 99%

See 1 more Smart Citation

Comparison of two crystal structures of TGF-β2: the accuracy of refined protein structures

Davies¹,

Schlunegger²,

Grütter³

1994

Acta Crystallogr D Biol Crystallogr

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Transforming growth factor-fl is a multifunctional cell-growth regulator and is a member of the TGF-fl superfamily of cytokines. Each monomer is 112 amino acids long and the mature active form is a 25 kDa homodimer. Recently, the crystal structure of TGF-fl2 has been determined independently in two laboratories [Daopin, Piez, Ogawa & Davies (1992). Science, 257, 369-373; Schlunegger & Grfitter (1992). Nature (London), 358,[430][431][432][433][434] and subsequently refined to higher resolutions [Daopin, Li & Davies (1993). Proteins Struct. Funct. Genet. In the press; Schlunegger & Griitter (1993). J. Mol. Biol. In the press]. A detailed structural comparison shows that the two structures are nearly identical with the differences mostly located on the mobile regions of the molecule. The r.m.s, differences between the two structures are 0.10 A for 104 pairs of C a atoms, 0.15 A for 434 pairs of main-chain atoms, 0.33 A for 860 out of 890 pairs of protein atoms and a correlation of 90% between the temperature B factors of all protein atoms. Based on a comparison of the water molecules, a B value of 60.0 A 2 is recommended as the cut off for modeling new waters. The structural identity is striking because in one case the material was expressed in vivo in CHO cells whereas in the other case it was expressed in E. coli and had to be refolded in vitro. The overall coordinate errors are estimated to be 0.21/~ from the Luzzati plot, 0.18 A from the O' A plot, 0.24 A with Cruickshank's equations and 0.25 A using the empirical method of Perry & Stroud. These estimates are comparable to the r.m.s, structure superposition. The r.m.s. differences correlate very well with the crystallographic B values and the relation is best described with the Cruickshank formula. In addition to the estimation of an overall error, a new application of the Cruickshank formula is presented here to estimate the local errors.

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Section: S Daopin D R Davies M P Schlunegger and M G Gristtermentioning

confidence: 87%

Section: Accuracy Assessmentsmentioning

confidence: 99%

Comparison of two crystal structures of TGF-β2: the accuracy of refined protein structures

Davies¹,

Schlunegger²,

Grütter³

1994

Acta Crystallogr D Biol Crystallogr

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“…TS provides the only de novo pathway to deoxythymidine monophosphate (dTMP) production and has been studied extensively from the standpoints of structure, function, and inhibition (Santi & Danenberg, 1984). The crystal structure, which has been determined to 2.3 A resolution for Lactobacillus casei TS (Hardy et al, 1987;Perry et al, 1990;Finer-Moore, unpubl. ), shows that the monomers form dimer contacts primarily between two, five-stranded beta sheets that maintain a unique +28" dihedral angle between strand directions (Kinemage 4).…”

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Reversible dissociation and unfolding of the dimeric protein thymidylate synthase

et al. 1992

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Conditions for in vitro unfolding and refolding of dimeric thymidylate synthase from Lactobacillus casei were found. Ultraviolet difference and circular dichroism spectra showed that the enzyme was completely unfolded at concentrations of urea over 5.5 M. As measured by restoration of enzyme activity, refolding was accomplished when 0.5 M potassium chloride was included in the refolding mixture. Recombination of subunits from catalytically inactive mutant homodimers to form an active hybrid dimer was achieved under these unfolding-refolding conditions, demonstrating a monomer to dimer association step.Keywords: dimerization; folding; oligomerization; thymidylate synthase Thymidylate synthase (TS) is a dimer of two identical, 35-kDa subunits (Kinemage 1) that catalyzes the conversion of deoxyuridine monophosphate (dUMP) and 5,lO-methylenetetrahydrofolate (CH2H4folate) to dTMP and 7,8-dihydrofolate (H2folate). TS provides the only de novo pathway to deoxythymidine monophosphate (dTMP) production and has been studied extensively from the standpoints of structure, function, and inhibition (Santi & Danenberg, 1984). The crystal structure, which has been determined to 2.3 A resolution for Lactobacillus casei TS (Hardy et al., 1987;Perry et al., 1990; Finer-Moore, unpubl.), shows that the monomers form dimer contacts primarily between two, five-stranded beta sheets that maintain a unique +28" dihedral angle between strand directions (Kinemage 4). Three of these strands form abeta kink that creates part of the active site pocket of the second monomer. Further, two active site arginine residues (178' and 179') are donated from the opposing monomer and coordinate with the phosphate moiety of the substrate (Kinemage 3). Thus, the dimeric structure of TS is essential for the function of the enzyme.In order to study interactions at the dimer interface of TS, we sought conditions that would allow the separation, unfolding, and reassembly of subunits. To date, there is no evidence that the dimer can be dissociated and reformed. In the present work, we show that TS can be denatured to unfolded monomers in urea and subsequently refolded to its catalytically active native state. Results and discussionThe concentration of urea required to unfold TS was determined using UV and CD spectroscopies. The difference in environment of tryptophan residues between native and unfolded protein was monitored by the UV absorbance change at 294 nm (Fig. 1). Three zones were apparent along the equilibrium unfolding transition: (1) a range below 3.5 M urea where spectral changes were not observed, (2) a sharp transition between 3.5 M and 5.5 M denaturant, and (3) a range above 5.5 M urea where no further change in absorbance was detected. The AGHZ0 calculated for this equilibrium unfolding transition at 0 M urea was 19.1 kcal mol" (see Materials and methods). To ascertain whether secondary structure was lost at high urea concentration, the CD spectrum of TS equilibrated in 8.0 M urea was compared with that of native protein (Fig. 2). With...

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“…Comparison of sequences of TS from ∼30 different organisms reveals that it is one of the most highly conserved enzymes (1,2). A large number of three-dimensional structures of free and ligand-bound TSs and TS mutants have been determined and provide a structural understanding of substrate recognition, stereochemical aspects of the reaction pathway (3), and roles of specific residues in the reaction chemistry (4).…”

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A Novel dCMP Methylase by Engineering Thymidylate Synthase

et al. 1997

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X-ray crystal structures of binary complexes of dUMP or dCMP with the Lactobacillus casei TS mutant N229D, a dCMP methylase, revealed that there is a steric clash between the 4-NH 2 of dCMP and His 199, a residue which normally H-bonds to the 4-O of dUMP but is not essential for activity. As a result, the cytosine moiety of dCMP is displaced from the active site and the catalytic thiol is moved from the C6 of the substrate about 0.5 Å further than in the wild-type TS-dUMP complex. We reasoned that combining the N229D mutation with mutations at residue 199 which did not impinge on the 4-NH 2 of dCMP should correct the displacements and further favor methylation of dCMP. We therefore prepared several TS N229D mutants and characterized their steady state kinetic parameters. TS H199A/N229D showed a 10 11 change in specificity for methylation of dCMP Versus dUMP. The structures of TS H199A/ N229D in complex with dCMP and dUMP confirmed that the position and orientation of bound dCMP closely approaches that of dUMP in wild-type TS, whereas dUMP was displaced from the optimal catalytic binding site.Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) by 5,10-methylene-5,6,7,8-tetrahydrofolate (CH 2 H 4 -folate) to form deoxythymidine-5′-monophosphate (dTMP) and 7,8-dihydrofolate (H 2 folate). Comparison of sequences of TS from ∼30 different organisms reveals that it is one of the most highly conserved enzymes (1, 2). A large number of three-dimensional structures of free and ligand-bound TSs and TS mutants have been determined and provide a structural understanding of substrate recognition, stereochemical aspects of the reaction pathway (3), and roles of specific residues in the reaction chemistry (4).Asn 229 is invariant in all TS's sequenced to date, and forms a cyclic hydrogen bond network with the 4-O and 3-NH of the substrate dUMP (Figure 2a) (5-7). Nevertheless, it can be replaced by numerous other residues, and the resultant mutants retain TS activity and also gain the capability to bind 2′-deoxycytidine 5′-monophosphate (dCMP) tightly (8,9). Moreover, alteration of Asn 229 to aspartate provides an enzyme which methylates dCMP more efficiently than dUMP, although the catalytic efficiency is still significantly lower than is the wild-type TS for its natural substrate dUMP (10, 11).

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Plastic adaptation toward mutations in proteins: Structural comparison of thymidylate synthases

Cited by 161 publications

References 50 publications

Comparison of two crystal structures of TGF-β2: the accuracy of refined protein structures

Comparison of two crystal structures of TGF-β2: the accuracy of refined protein structures

Reversible dissociation and unfolding of the dimeric protein thymidylate synthase

A Novel dCMP Methylase by Engineering Thymidylate Synthase

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