Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase.

The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474-11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed.

Modeling, mutagenesis, and structural studies on the fully conserved phosphate-binding loop (loop 8) of triosephosphate isomerase: toward a new substrate specificity.

Loop 8 (residues 232-242) in triosephosphate isomerase (TIM) is a highly conserved loop that forms a tight binding pocket for the phosphate moiety of the substrate. Its sequence includes the fully conserved, solvent-exposed Leu238. The tight phosphate-binding pocket explains the high substrate specificity of TIM being limited to the in vivo substrates dihydroxyacetone-phosphate and D-glyceraldehyde-3-phosphate. Here we use the monomeric variant of trypanosomal TIM for exploring the structural consequences of shortening this loop. The mutagenesis, guided by extensive modeling calculations and followed up by crystallographic characterization, is aimed at widening the phosphate-binding pocket and, consequently, changing the substrate specificity. Two new variants were characterized. The crystal structures of these variants indicate that in monomeric forms of TIM, the Leu238 side-chain is nicely buried in a hydrophobic cluster. Monomeric forms of wild-type dimeric TIM are known to exist transiently as folding intermediates; our structural analysis suggests that in this monomeric form, Leu238 of loop 8 also adopts this completely buried conformation, which explains its full conservation across the evolution. The much wider phosphate-binding pocket of the new variant allows for the development of a new TIM variant with a different substrate specificity.

The ionization of a buried glutamic acid is thermodynamically linked to the stability of Leishmania mexicana triose phosphate isomerase.

The amino acid sequence of Leishmania mexicana triose phosphate isomerase is unique in having at position 65 a glutamic acid instead of a glutamine. The stability properties of LmTIM and the E65Q mutant were investigated by pH and guanidinium chloride-induced unfolding. The crystal structure of E65Q was determined. Three important observations were made: (a) there are no structural rearrangements as the result of the substitution; (b) the mutant is more stable than the wild-type; and (c) the stability of the wild-type enzyme shows strong pH dependence, which can be attributed to the ionization of Glu65. Burying of the Glu65 side chain in the uncharged environment of the dimer interface results in a shift in pKa of more than 3 units. The pH-dependent decrease in overall stability is due to weakening of the monomer-monomer interactions (in the dimer). The E65Q substitution causes an increase in stability as the result of the formation of an additional hydrogen bond in each subunit (DeltaDeltaG degrees of 2 kcal.mol-1 per monomer) and the elimination of a charged group in the dimer interface (DeltaDeltaG degrees of at least 9 kcal.mol-1 per dimer). The computated shift in pKa and the stability of the dimer calculated from the charge distribution in the protein structure agree closely with the experimental results. The guanidinium chloride dependence of the unfolding constant was smaller than expected from studies involving monomeric model proteins. No intermediates could be identified in the unfolding equilibrium by combining fluorescence and CD measurements. Study of a stable monomeric triose phosphate isomerase variant confirmed that the phenomenon persists in the monomer.

The X-ray structure of a mutant eye lens beta B2-crystallin with truncated sequence extensions.

beta-Crystallins are oligomeric eye lens proteins that are related to monomeric gamma-crystallins by domain swapping: like gamma-crystallins, they are comprised of two similar domains but they differ in having long sequence extensions. beta B2, a major component of beta-crystallin oligomers, self-associates to a homodimer in solution. In two crystal structures of native beta B2, the protein is a 222-symmetric tetramer of eight domains. It has previously been shown that a mutant of rat beta B2-crystallin, in which the bulk of the N- and C-terminal sequence extensions has been deleted, assembles into dimers and tetramers. Here we present the 3.0 A resolution X-ray structure of the tetramer, beta B2 delta NC1. The mutant tetramer has a very similar set of domain interactions to the native structure. However, the structures differ in the relative orientation of the two sets of four domains. The paired N- and C-terminal domain interface, which is at the heart of the dimer structure, is very similar to the native structure. However, the truncation of the C-terminal extension removes an important tryptophan residue, which prevents the extension from acting as a (non-covalent) linker, as it does in native beta B2. There is a knock-on structural effect that removes a contact between extension and covalent linker, and this appears to cause a small twist in the linker that is amplified into a 20 degrees rotation between sets of paired domains.

Towards a molecular understanding of phase separation in the lens: a comparison of the X-ray structures of two high Tc gamma-crystallins, gammaE and gammaF, with two low Tc gamma-crystallins, gammaB and gammaD.

gamma-Crystallins, although closely related in sequence, show intriguing differences in their temperature-dependent interactions: those that have a high or intermediate Tc for phase separation are cryoproteins whereas low Tc gamma-crystallins are not. To address the molecular basis of phase separation, X-ray crystallography has been used to define the structural differences between high and low Tc gamma-crystallins. A pre-requisite for this study was to clarify the assignment of bovine gene sequences to bovine gamma-crystallin proteins used for biophysical measurements. Based on nucleotide sequence analyses of gamma E and gamma F bovine crystallin genes, gamma F corresponds to the previously crystallised high Tc protein bovine gamma IVa and gamma E corresponds to the high Tc bovine protein fraction previously known as gamma IIIa. The gamma F sequence has enabled the completion of the refinement of the bovine gamma F crystal structure which shows that the molecule has an additional surface tryptophan explaining why gamma F has different spectroscopic properties from gamma B. A high Tc protein from rat lens, gamma E crystallin, has been crystallised and the X-ray structure solved at 2.3 A resolution. Comparison of the X-ray structures of two high Tc proteins, rat gamma E and bovine gamma F, with the structures of two low Tc proteins, bovine gamma B and bovine gamma D, shows that the main conformational change between high and low Tc proteins is in the cd surface loop of motif 3. All four structures have numerous ion pairs on their surfaces leading to a high surface charge density, yet with low overall charge. Comparison of the lattice contacts of the two high Tc proteins with the two low Tc gamma-crystallins indicates that these high Tc proteins utilise more amino-aromatic interactions such as between histidine and arginine. Comparison of the sequences of all the gamma-crystallins which have been characterised for phase separation temperature indicates that only residue Arg/Lys 163 uniquely distinguishes cryo from non-cryo gamma-crystallins and it is close to the altered surface loop. Although this region probably contributes to phase separation, Tc is likely to be a function of an overall global property that is responsive to overall charge distribution. Calculated dipole moments of native gamma-crystallins, low Tc gamma-crystallin sequences threaded into high Tc gamma-crystallin structures, and vice versa, show how both sequence and 3D structure contribute to this overall property. High Tc gamma-crystallins have on average higher Arg/Lys ratios and higher histidine content. It is hypothesised that this increases the proportion of surface static paired charged networks which thus reduces the repulsive hydration force and so increases the attractive interactions of the protein-rich phase in binary liquid phase separation.

The X-ray structures of two mutant crystallin domains shed light on the evolution of multi-domain proteins.

We use protein engineering and crystallography to simulate aspects of the early evolution of beta gamma-crystallins by observing how a single domain oligomerizes in response to changes in a sequence extension. The crystal structure of the C-terminal domain of gamma beta-crystallin with its four-residue C-terminal extension shows that the domain does not form a symmetric homodimer analogous to the two-domain pairing in beta gamma-crystallins. Instead the C-terminal extension now forms heterologous interactions with other domains leading to the solvent exposure of the natural hydrophobic interface with a consequent loss in protein solubility. However, this domain truncated by just the C-terminal tyrosine forms a symmetric homodimer of domains in the crystal lattice.