Exploration of Natural and Artificial Sequence Spaces: Towards a Functional Remodeling of Membrane-bound Cytochrome P450

Abstract

Two complementary methods are described that associate in vitro and in vivo steps to generate sequence diversity by segment directed saturated mutagenesis and family shuffling. A high-throughput DNA chip-based procedure for the characterization and potentially the equalization of combinatorial libraries is also presented. Using these approaches, two combinatorial libraries of cytochrome P450 variants derived from the CYP1A subfamily were constructed and their sequence diversity characterized. The results of functional screening using high-throughput tools for the characterization of membrane P450-catalyzed activities, suggest that the 204–214 sequence segment of human CYP1A1 is not critical for polycyclic aromatic hydrocarbon recognition, as was hypothesized from previous data. Moreover, mutations in this segment do not alter the discrimination between alkoxyresorufins, which, for all tested mutants, remained similar to that of wild-type CYP1A1. In contrast, the constructed CYP1A1–CYP1A2 mosaic structures, containing multiple crossovers, exhibit a wide range of substrate preference and regioselectivity. These mosaic structures also discriminate between closely related alkoxyresorufin substrates. These results open the way to global high-throughput analysis of structure–function relationships using combinatorial libraries of enzymes together with libraries of structurally related substrates.     

Computational methods for sequence mapping of large combinatorial libraries and deduced sequence signatures

Here we describe a computational approach for the high-throughput sequence mapping of combinatorial libraries obtained by DNA shuffling. Original algorithms and their software implementation were developed for the automated and reliable analysis of hybridization data of differentially labeled oligonucleotide probes with PCR products spotted on DNA microarrays. This novel approach allows a context-dependent sequence attribution tolerant to fluctuations in experimental conditions and is well adapted to hybridization signals of variable qualities resulting from high-throughput PCR amplification from colonies. In addition, the analysis permits the calculation of sequence signatures that are characteristic of combinatorial library structure, defects, and diversity. The approach is of interest for the characterization and the equalization (library reduction to nonredundant structures) of combinatorial libraries involved in directed evolution and could be extrapolated to high-throughput polymorphism analysis.     

Microarray-based method for combinatorial library sequence mapping and characterization

Here we describe a DNA-chip-based method for high-throughput sequence mapping. This involves competitive hybridization between short and differentially labeled fluorescent oligonucleotide probes and glass-supported PCR products. Competition between an excess of oligonucleotide probes targeting the same sequence segment improves sequence discrimination and reduces sensitivity to experimental conditions such as probe concentrations, hybridization, and washing temperatures and durations. The method was found to be particularly adapted to sequence mapping of combinatorial libraries obtained by DNA shuffling between members of a gene family. We present an application of this technique for the characterization of recombination biases in combinatorial libraries used in directed evolution.     

A combinatorial approach to substrate discrimination in the P450 CYP1A subfamily

A comparison of all known mammalian CYP1A sequences identifies nineteen sequence regions that are conserved within all 1A1s or within all 1A2s but at the same time systematically differ between any 1A1 and any 1A2. The purpose of this study was to explore links between these specific CYP1A sequence signatures and substrate specificity shift through the kinetic analysis of combinatorial variants of increasing complexity. The less complex variants correspond to multiple mutations within a short segment of their sequence. The more complex variants correspond to mosaic P450s recombining 1A1 and 1A2 sequences (up to 5 crossovers per sequence). Fifty-eight such functional CYP1A variants and parental wild-type enzymes were expressed in yeast and assayed with 7-alkoxyresorufins and ethoxyflurorescein ethyl ester as substrates. Observed kinetic data were analyzed by multivariate statistical analyses and hierarchical clustering in order to highlight correlations and identify potential sequence-activity relationships within the three-dimensional function space investigated. Several variants are outliers in these representations and show a redistribution of their substrate specificity compared to wild-type CYP1As. Some combinations of sequence elements were identified that significantly discriminate between 1A1 and 1A2 for these three substrates. The comparison of this combinatorial approach with previous results of site-directed mutagenesis is discussed.     

In vitro enzyme evolution: the screening challenge of isolating the one in a million

The generation of large mutant libraries for in vitro enzyme evolution presents the challenge of effectively screening libraries of 10⁴–10⁷ mutants on the basis of simultaneously assaying their biocatalytic activity. In this review, we highlight the main steps involved in this process, describe the alternative approaches to address this challenge, survey the state-of-the-art technology and assess achievements already made. It is anticipated that, as a result of the expected accomplishment of further improvements in high-throughput screening that will allow routine screening of whole libraries, the number of useful new and improved enzymes derived through in vitro enzyme evolution will expand rapidly in the near future.     

Quantitative assessment of peptide sequence diversity in M13 combinatorial peptide phage display libraries

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0+/-1.6% of the random dodecapeptides and 7.9+/-2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a beta-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

High-throughput characterization and quality control of small-molecule combinatorial libraries

To fully realize the potential of combinatorial synthesis and high-throughput screening for increasing the efficiency of the drug discovery and development process, issues related to compound purity must be addressed. Impurities, often present after synthesis, can lead to ambiguous screening results and inhibit the development of quality structure-activity relationships. The demand for high-throughput analytical characterization of combinatorial libraries has prompted the development of more rapid methods to keep pace with compound production. Recent progress has focused upon the development of parallel separation methods, multiplexed detector interfaces, and synergistic combinations of different detectors possessing complementary selectivities.     

Determination of the substrate specificity of tripeptidyl-peptidase I using combinatorial peptide libraries and development of improved fluorogenic substrates

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant kcat/KM. In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1' and P2' residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.     

Common active site architecture and binding strategy of four phenylpropanoid P450s from Arabidopsis thaliana as revealed by molecular modeling

Despite extensive primary sequence diversity, crystal structures of several bacterial cytochrome P450 monooxygenases (P450s) and a single eukaryotic P450 indicate that these enzymes share a structural core of alpha-helices and beta-sheets and vary in the loop regions contacting individual substrates. To determine the extent to which individual structural features are conserved among divergent P450s existing in a single biosynthetic pathway, we have modeled the structures of four highly divergent P450s (CYP73A5, CYP84A1, CYP75B1, CYP98A3) in the Arabidopsis phenylpropanoid pathway synthesizing lignins, flavonoids and anthocyanins. Analysis of these models has indicated that, despite primary sequence identities as low as 13%, the structural cores and several loop regions of these P450s are highly conserved. Substrate docking indicated that all four enzymes employ a common strategy to identify their substrates in that their cinnamate-derived substrates align along helix I with their aromatic ring positioned towards the C-terminus of this helix and their aliphatic tails positioned towards the N-terminus. Further similarity was observed in the way the substrates contact the consensus P450 substrate recognition sites (SRS). Residues predicted to contact the aromatic ring region exist in SRS5, SRS6 and the C-terminal portion of SRS4 and residues contacting the distal end of each substrate exist in SRS1, SRS2 and the N-terminal portion of SRS4. Alignments of the regions contacting the aromatic ring region indicate that SRS4, SRS5 and SRS6 share higher degrees of sequence conservation than found in SRS1, SRS2 or the full-length protein.     

High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2

The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 ± 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure–function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.     

Directed evolution of sulfotransferases and paraoxonases by ancestral libraries

Large libraries of randomly mutated genes are applied in directed evolution experiments in order to obtain sufficient variability. These libraries, however, contain mostly inactive variants, and the very low frequency of improved variants can only be isolated by high-throughput screening. Small but efficient libraries comprise an attractive alternative. Here, we describe the application of ancestral libraries—libraries based on mutations predicted by phylogenetic analysis and ancestral inference. We designed and constructed such libraries using serum paraoxonases and cytosolic sulfotransferases (SULTs) as model enzymes. Both of these enzyme families exhibit a range of activities in drug metabolism and detoxification of xenobiotics. The ancestral serum paraoxonase and SULT libraries were screened by low-throughput means, including HPLC, using substrates and/or reactions with which all family members exhibit low activity. The libraries showed a remarkably high frequency of highly polymorphic and functionally diverse variants. Screening of as few as 300 variants enabled the isolation of mutants with up to 50-fold higher activity than the starting point enzyme. Structural and kinetic characterizations of an evolved SULT variant show how few ancestral mutations reshaped the active site and modulated the enzyme's specificity. Ancestral libraries therefore comprise a means of focusing diversity to positions and mutations that readily trigger changes in substrate and/or reaction specificity, thereby facilitating the isolation of new enzyme variants for a variety of different substrates and reactions by medium-throughput or even low-throughput screens.     

Therapeutic peptides

Novel peptide therapeutics are increasingly making their way into clinical application. Indeed, certain naturally derived peptides have been successful drugs for many years. With the advent of large biological and synthetic peptide libraries and high-throughput screening, many promising candidates could soon be added to the list of peptides under development. These advances have introduced new strategies for the administration of peptide drugs and improvements of clearance half-lives in vivo. Despite the potential obstacles that remain, peptide therapeutics are poised to play a significant role in the treatment of diseases ranging from Alzheimer's disease to cancer.     

Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration to guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17-hydroxylase and 21-hydroxylase activities were decreased to 20–25% of control values by the higher dose of ABT. Mitochondrial 11β-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3β-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.     

Contributions of CDR3 to V H H domain stability and the design of monobody scaffolds for naive antibody libraries

Camelids produce functional antibodies devoid of light chains. Autonomous heavy chain variable (V(H)H) domains in these molecules have adapted to the absence of the light chain in the following ways: bulky hydrophobic residues replace small aliphatic residues in the former light chain interface, and residues from the third complementarity-determining region (CDR3) pack against the framework and stabilize the global V(H)H domain fold. To determine the specific roles of CDR3 residues in framework stabilization, we used nai;ve phage-displayed libraries, combinatorial alanine-scanning mutagenesis and biophysical characterization of purified proteins. Our results indicate that in the most stable scaffolds, the structural residues in CDR3 reside near the boundaries of the loop and pack against the framework to form a small hydrophobic core. These results allow us to differentiate between structural CDR3 residues that should remain fixed, and CDR3 residues that are tolerant to substitution and can therefore be varied to generate functional diversity within phage-displayed libraries. These methods and insights can be applied to the rapid design of heavy chain scaffolds for the identification of novel ligands using synthetic, antibody-phage libraries. In addition, they shed light on the relationships between CDR3 sequence diversity and the structural stability of the V(H)H domain fold.     

Factors important in evolutionary shaping of immunoglobulin gene loci

Background

The extraordinary diversity characterizing the antibody repertoire is generated by both evolution and lymphocyte development. Much of this diversity is due to the existence of immunoglobulin (Ig) variable region gene segment libraries, which were diversified during evolution and, in higher vertebrates, are used in generating the combinatorial diversity of antibody genes. The aim of the present study was to address the following questions: What evolutionary parameters affect the size and structure of gene libraries? Are the number of genes in libraries of contemporary species, and the corresponding gene locus structure, a random result of evolutionary history, or have these properties been optimized with respect to individual or population fitness? If a larger number of genes or different genome structures do not increase the fitness, then the current structure is probably optimized.

Results

We used a simulation of variable region gene library evolution. We measured the effect of different parameters on gene library size and diversity, and the corresponding fitness. We found compensating relationships between parameters, which optimized Ig library size and diversity.

Conclusions

We conclude that contemporary species' Ig libraries have been optimized by evolution in terms of Ig sequence lengths, the number and diversity of Ig genes, and antibody-antigen affinities.     

Protein engineering by cDNA recombination in yeasts: shuffling of mammalian cytochrome P-450 functions

We have constructed, in the yeast Saccharomyces cerevisiae, a mosaic assembly of genes by in vivo recombination of partially homologous sequences. The approach was tested on cDNAs encoding functionally distinct mammalian cytochromes P-450 (P-450). The selection for recombinant cDNAs used the transformation of yeast cells, which required the recircularization of a linearized plasmid by recombination of two partially homologous cDNAs. Libraries of mosaic genes with bipartite or tripartite structures were generated by intramolecular and intermolecular recombination events. The presence of yeast promoter and terminator sequences on the flanking sides of the recombined cDNAs has allowed the synthesis of encoded mosaic proteins. A library of yeast clones producing recombinant mouse P-450 P1 and rabbit P-450 LM4 was screened using functional criteria to identify chimeras with shuffled substrate specificity. Restriction mapping of mosaic genes, biochemical analysis of the synthesized proteins, comparison of chimeric enzymes, and the alignment of sequences with bacterial P-450 camphor hydroxylase of known three-dimensional structure, all suggest that the P-450 P1 amino acid residues 203-238 play a major role in the control of cytochrome activity toward carcinogenic polycyclic aromatic hydrocarbons. Similar approaches to structure-function analysis are believed to be applicable to other protein families.     

Optimization of combinatorial mutagenesis

Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (e.g., stability, activity, immunogenicity). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries--a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 10? variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and novelty as well as library construction technique, and identify optimal libraries for experimental evaluation.