Protein Engineering Design and Selection - current issue

Structural insight into the binding mode between the targeting domain of ALE-1 (92AA) and pentaglycine of peptidoglycan

Hirakawa, H., Akita, H., Fujiwara, T., Sugai, M., Kuhara, S. — 2009-06-19

ALE-1 is a glycylglycine endopeptidase that selectively targets and lyses Staphylococcus aureus, and is expected to be a next generation antibacterial agent because of its substrate specificity to pathogenic bacteria. It has a central catalytic domain and a targeting domain called 92AA. 92AA has been shown to recognize pentaglycine, but the molecular mechanism by which it recognizes and interacts with pentaglycine has not been elucidated. To predict the binding modes of pentaglycine is important for estimating the catalytic reaction mechanism of ALE-1. In the present study, we characterized the binding cleft of 92AA by a computational method and modeled the complexes formed between 92AA and the pentaglycine of peptidoglycan by a binding simulation. In addition, we performed precise simulations of the molecular dynamics by which the complexes identify the amino acid residues interacting with the pentaglycine. We also experimentally constructed mutants in which the amino acid residues present in the binding cleft were changed by site-directed mutagenesis and assessed their ability to bind to peptidoglycan by ELISA. Based on the results of these analyses, we proposed a mode of binding between 92AA and the pentaglycine of peptidoglycan, and modeled the energetically stable complexes between 92AA and the pentaglycine.

Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase

2009-06-19

Disaccharide phosphorylases are interesting enzymes for the production of sugar phosphates from cheap starting materials and for the synthesis of novel glycosides. Cellobiose phosphorylase (CP) from Cellulomonas uda was subjected to directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase (LP) activity, useful for the production of -d-galactose 1-phosphate. In a first round, random mutagenesis was performed on part of the CP gene and the resultant library was selected on minimal lactose medium. One clone containing six amino acid mutations was found with increased LP activity compared with the wild-type CP enzyme. The negative and neutral mutations were eliminated by site-directed mutagenesis and the resultant enzyme variant containing two amino acid substitutions (T508A/N667T) showed more LP activity than the parent mutant. Saturation mutagenesis of the beneficial sites and screening for improved mutants allowed us to identify the T508I/N667A mutant which has 7.5 times higher specific activity on lactose than the wild-type. The kinetic parameters of the mutants were determined and showed that the increased LP activity was caused by a higher k_cat value. This is the first report of an engineered CP with modified substrate specificity.

Distributions of enzyme residues yielding mutants with improved substrate specificities from two different directed evolution strategies

Paramesvaran, J., Hibbert, E. G., Russell, A. J., Dalby, P. A. — 2009-06-19

A previous study of random mutations, mostly introduced by error-prone PCR (EPPCR) or DNA shuffling (DS), demonstrated that those closer to the enzyme active site were more effective than distant ones at improving enzyme activity, substrate specificity or enantioselectivity. Since then, many studies have taken advantage of this observation by targeting site-directed saturation mutagenesis (SDSM) to residues closer to or within enzyme active sites. Here, we have analysed a set of SDSM studies, in parallel to a similar set from EPPCR/DS, to determine whether the greater range of amino-acid types accessible by SDSM affects the distances at which the most effective sites occur. We have also analysed the relative effectiveness for obtaining beneficial mutants of residues with different degrees of natural sequence variation, as determined by their sequence entropy which is related to sequence conservation. These analyses attempt to answer the question—how well focused have targeted mutagenesis strategies been? We also compared two different sets of active-site atoms from which to measure distances and found that the inclusion of catalytic, substrate and cofactor atoms refined the analysis compared to using a single key catalytic atom. Using this definition, we found that EPPCR/DS is not effective for altering substrate specificity at sites that are within 5 Å of the active-site atoms. In contrast, SDSM is most effective when targeted to residues at <5–6 Å from the catalytic, substrate or cofactor atom, and also for residues with intermediate sequence entropies. Furthermore, SDSM is capable of altering substrate specificity at highly and completely conserved residues in the active site. The results suggest ways in which directed evolution by SDSM could be improved for greater efficiency in terms of reducing the library sizes required to obtain beneficial mutations that alter substrate specificity.

Directed evolution of Candida antarctica lipase A using an episomaly replicating yeast plasmid

Sandstrom, A. G., Engstrom, K., Nyhlen, J., Kasrayan, A., Backvall, J.-E. — 2009-06-19

We herein report the first directed evolution of Candida antarctica lipase A (CalA), employing a combinatorial active-site saturation test (CAST). Wild-type CalA has a modest E-value of 5.1 in kinetic resolution of 4-nitrophenyl 2-methylheptanoate. Enzyme variants were expressed in Pichia pastoris by using the episomal vector pBGP1 which allowed efficient secretory expression of the lipase. Iterative rounds of CASTing yielded variants with good selectivity toward both the (S)- and the (R)-enantiomer. The best obtained enzyme variants had E-values of 52 (S) and 27 (R).

Stabilising the DNA-binding domain of p53 by rational design of its hydrophobic core

Khoo, K. H., Joerger, A. C., Freund, S. M.V., Fersht, A. R. — 2009-06-19

The core domain of the tumour suppressor p53 is of inherently low thermodynamic stability and also low kinetic stability, which leads to rapid irreversible denaturation. Some oncogenic mutations of p53 act by just making the core domain thermosensitive, and so it is the target of novel anti-cancer drugs that bind to and stabilise the protein. Increasing the stability of the unstable core domain has also been crucial for biophysical and structural studies, in which a stabilised quadruple mutant (QM) is currently used. We generated an even more stabilised hexamutant (HM) by making two additional substitutions, Y236F and T253I, to the QM. The residues are found in the more stable paralogs p63 and p73 and stabilise the wild-type p53 core domain. We solved the structure of the HM core domain by X-ray crystallography at 1.75 Å resolution. It has minimal structural changes from QM that affect the packing of hydrophobic core residues of the β-sandwich. The full-length HM was also fully functional in DNA binding. HM was more stable than QM at 37°C. Anomalies in biophysics and spectroscopy in urea-mediated denaturation curves of HM implied the accumulation of a folding intermediate, which may be related to those detected in kinetic experiments. The two additional mutations over-stabilise an unfolding intermediate. These results should be taken into consideration in drug design strategies for increasing the stability of temperature-sensitive mutants of p53.

A structural model for the HAT domain of Utp6 incorporating bioinformatics and genetics

Champion, E. A., Kundrat, L., Regan, L., Baserga, S. J. — 2009-06-19

The half-a-tetratricopeptide (HAT) repeat motif is of interest because it is found exclusively in proteins that are involved in RNA metabolism. Little is known about structure–function relationships in this class of repeat motif. Here, we present the results of a combined bioinformatics, modeling and mutagenesis study of the HAT domain of Utp6. We have derived a new HAT consensus, delineated its structure-defining residues and, by homology modeling, have placed these residues in a structural context. By considering only HAT motifs from Utp6 proteins, we identified residues that are shared by, and unique to, only this subset of HAT motifs, suggesting a key functional role. Employing both random and directed mutagenesis of the HAT domain in yeast Utp6, we have identified residues whose mutation results in loss of function. By examining these residues in the context of the homology model, we have delineated those that act by perturbing structure and those that more likely have a direct effect on function. Importantly, the residues we predict to have a direct effect on function map together on the tertiary structure, thus defining a potential functional interaction surface.

Prediction and classification of chemokines and their receptors

Lata, S., Raghava, G.P.S. — 2009-06-19

Chemokines are low molecular mass cytokine-like proteins that orchestrate myriads of immune functions like leukocyte trafficking, T cell differentiation, angiogenesis, hematopeosis and mast cell degranulation. Chemokines also play a role as HIV-1 inhibitor and act as potent natural adjuvant in antitumor immunotherapy. Receptors for these molecules are all seven-pass transmembrane G-protein-coupled receptors that are intimately involved with chemokines in a wide array of physiological and pathological conditions. These receptors also have a major role as co-receptors for HIV-1 entry into target cells. Therefore, chemokine receptors have proven to be excellent targets for small molecule in pharmaceutical industry. The immense importance of chemokines and their receptors motivated us to develop a support vector machine-based method ChemoPred to predict this important class of proteins and further classify them into subfamilies. ChemoPred is capable of predicting chemokines and chemokine receptors with an accuracy of 95.08% and 92.19%, respectively. The overall accuracy of classification of chemokines into three subfamilies was 96.00% and that of chemokine receptors into three families was 92.87%. The server ChemoPred is freely available at www.imtech.res.in/raghava/chemopred.