Single-site enzymatic cleavage of yeast genomic DNA mediated by triple helix formation.

Physical mapping of chromosomes would be facilitated by methods of breaking large DNA into manageable fragments, or cutting uniquely at genetic markers of interest. Key issues in the design of sequence-specific DNA cleaving reagents are the specificity of binding, the generalizability of the recognition motif, and the cleavage yield. Oligonucleotide-directed triple helix formation is a generalizable motif for specific binding to sequences longer than 12 base pairs within DNA of high complexity. Studies with plasmid DNA show that triple helix formation can limit the operational specificity of restriction enzymes to endonuclease recognition sequences that overlap oligonucleotide-binding sites. Triple helix formation, followed by methylase protection, triple helix-disruption, and restriction endonuclease digestion produces near quantitative cleavage at the single overlapping triple helix-endonuclease site. As a demonstration that this technique may be applicable to the orchestrated cleavage of large genomic DNA, we report the near quantitative single-site enzymatic cleavage of the Saccharomyces cerevisiae genome mediated by triple helix formation. The 340-kilobase yeast chromosome III was cut uniquely at an overlapping homopurine-EcoRI target site 27 base pairs long to produce two expected cleavage products of 110 and 230 kilobases. No cleavage of any other chromosome was detected. The potential generalizability of this technique, which is capable of near quantitative cleavage at a single site in at least 14 megabase pairs of DNA, could enable selected regions of chromosomal DNA to be isolated without extensive screening of genomic libraries.     

Designed divergent evolution of enzyme function

It is generally believed that proteins with promiscuous functions divergently evolved to acquire higher specificity and activity, and that this process was highly dependent on the ability of proteins to alter their functions with a small number of amino acid substitutions (plasticity). The application of this theory of divergent molecular evolution to promiscuous enzymes may allow us to design enzymes with more specificity and higher activity. Many structural and biochemical analyses have identified the active or binding site residues important for functional plasticity (plasticity residues). To understand how these residues contribute to molecular evolution, and thereby formulate a design methodology, plasticity residues were probed in the active site of the promiscuous sesquiterpene synthase gamma-humulene synthase. Identified plasticity residues were systematically recombined based on a mathematical model in order to construct novel terpene synthases, each catalysing the synthesis of one or a few very different sesquiterpenes. Here we present the construction of seven specific and active synthases that use different reaction pathways to produce the specific and very different products. Creation of these enzymes demonstrates the feasibility of exploiting the underlying evolvability of this scaffold, and provides evidence that rational approaches based on these ideas are useful for enzyme design.     

Cleavage of pre-tRNAs by the splicing endonuclease requires a composite active site

Splicing is required for the removal of introns from a subset of transfer RNAs in all eukaryotic organisms. The first step of splicing, intron recognition and cleavage, is performed by the tRNA-splicing endonuclease, a tetrameric enzyme composed of the protein subunits Sen54, Sen2, Sen34 and Sen15. It has previously been demonstrated that the active sites for cleavage at the 5' and 3' splice sites of precursor tRNA are contained within Sen2 and Sen34, respectively. A recent structure of an archaeal endonuclease complexed with a bulge-helix-bulge RNA has led to the unexpected hypothesis that catalysis requires a critical 'cation-pi sandwich' composed of two arginine residues that serve to position the RNA substrate within the active site. This motif is derived from a cross-subunit interaction between the two catalytic subunits. Here we test the role of this interaction within the eukaryotic endonuclease and show that catalysis at the 5' splice site requires the conserved cation-pi sandwich derived from the Sen34 subunit in addition to the catalytic triad of Sen2. The catalysis of pre-tRNA by the eukaryotic tRNA-splicing endonuclease therefore requires a previously unrecognized composite active site.     

计算机辅助蛋白结构预测及酶的计算设计研究进展

随着不可再生资源的日益减少以及化学催化所带来的环境污染问题的日益严重,应用低污染的生物催化技术改造或取代传统化工工艺已经成为可持续发展的研究热点。酶作为一种重要的生物催化剂,具有高效无污染、专一性较强等优点,在化工、医药等领域具有巨大的应用潜力和良好的发展前景。但由于新功能酶的开发速度较慢,使其应用受到严重限制。酶的理性设计是新酶发现的一个重要来源,随着计算机技术的发展,蛋白质结构预测方法和新功能酶计算设计策略得到迅猛的发展,已成为酶功能改造的有力工具和新的研究前沿。本文就蛋白质结构预测方法、新功能酶计算设计方法和策略以及未来发展趋势进行简要介绍和讨论。     

Contributions of hydrogen bonds of Thr 157 to the thermodynamic stability of phage T4 lysozyme

Measurements of changes in structure and stability caused by 13 different substitutions for threonine 157 in phage T4 lysozyme show that the most stable lysozyme variants contain hydrogen bonds analogous to those in the wild-type enzyme and that structural adjustments allow the protein to be surprisingly tolerant of amino-acid substitutions.     

Computational design of receptor and sensor proteins with novel functions

The formation of complexes between proteins and ligands is fundamental to biological processes at the molecular level. Manipulation of molecular recognition between ligands and proteins is therefore important for basic biological studies and has many biotechnological applications, including the construction of enzymes, biosensors, genetic circuits, signal transduction pathways and chiral separations. The systematic manipulation of binding sites remains a major challenge. Computational design offers enormous generality for engineering protein structure and function. Here we present a structure-based computational method that can drastically redesign protein ligand-binding specificities. This method was used to construct soluble receptors that bind trinitrotoluene, l-lactate or serotonin with high selectivity and affinity. These engineered receptors can function as biosensors for their new ligands; we also incorporated them into synthetic bacterial signal transduction pathways, regulating gene expression in response to extracellular trinitrotoluene or l-lactate. The use of various ligands and proteins shows that a high degree of control over biomolecular recognition has been established computationally. The biological and biosensing activities of the designed receptors illustrate potential applications of computational design.     

Detection of single base substitutions in total genomic DNA

Certain single base substitutions causing genetic diseases or resulting in polymorphisms linked to mutant alleles, alter a restriction enzyme cleavage site and can therefore be detected in total genomic DNA using DNA blots. Many base substitutions do not lead to an altered restriction site, but these can be detected using synthetic oligonucleotides as hybridization probes if the DNA sequence surrounding the base substitution is known. In the case of beta-thalassaemia, where 22 different single base mutations have been identified, a large number of probes would be required for diagnosis. An approach which was used to detect mutations in viral DNA involves the S1 nuclease treatment of heteroduplexes formed between wild-type and mutant DNA. Although certain single base mismatches are cleaved by S1 nuclease (ref. 11 and T. Shenk, personal communication), many other mismatches examined by this procedure are not cleaved (B. Seed, personal communication; R.M.M., unpublished data). Heteroduplexes between mutant and wild-type subgenomic fragments of double-stranded reovirus RNA migrate slower than the corresponding homoduplexes in polyacrylamide gels containing 7 M urea, but it is not known whether this method is applicable to DNA heteroduplexes containing single base mismatches. Here we describe a procedure that involves the electrophoretic separation of DNA heteroduplexes in a well-characterized gel system. We show that four different human beta-thalassaemia alleles with known single base mutations can be detected with as little as 5 micrograms of total genomic DNA. The method should be useful in the localization and diagnosis of mutations associated with genetic diseases.     

Prediction of the structure of a receptor-protein complex using a binary docking method.

To validate procedures of rational drug design, it is important to develop computational methods that predict binding sites between a protein and a ligand molecule. Many small molecules have been tested using such programs, but examination of protein-protein and peptide-protein interactions has been sparse. We were able to test such applications once the structures of both the maltose-binding protein (MBP) and the ligand-binding domain of the aspartate receptor, which binds MBP, became available. Here we predict the binding site of MBP to its receptor using a 'binary docking' technique in which two MBP octapeptide sequences containing mutations that eliminate maltose chemotaxis are independently docked to the receptor. The peptides in the docked solutions superimpose on their original positions in the structure of MBP and allow the formation of an MBP-receptor complex. The consistency of the computational and biological results supports this approach for predicting protein-protein and peptide-protein interactions.     

Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease

Most eukaryotic messenger RNA precursors (pre-mRNAs) undergo extensive maturational processing, including cleavage and polyadenylation at the 3'-end. Despite the characterization of many proteins that are required for the cleavage reaction, the identity of the endonuclease is not known. Recent analyses indicated that the 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF-73) might be the endonuclease for this and related reactions, although no direct data confirmed this. Here we report the crystal structures of human CPSF-73 at 2.1 A resolution, complexed with zinc ions and a sulphate that might mimic the phosphate group of the substrate, and the related yeast protein CPSF-100 (Ydh1) at 2.5 A resolution. Both CPSF-73 and CPSF-100 contain two domains, a metallo-beta-lactamase domain and a novel beta-CASP (named for metallo-beta-lactamase, CPSF, Artemis, Snm1, Pso2) domain. The active site of CPSF-73, with two zinc ions, is located at the interface of the two domains. Purified recombinant CPSF-73 possesses RNA endonuclease activity, and mutations that disrupt zinc binding in the active site abolish this activity. Our studies provide the first direct experimental evidence that CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.     

A mutant T4 lysozyme displays five different crystal conformations

Phage T4 lysozyme consists of two domains between which is formed the active-site cleft of the enzyme. The crystallographically determined thermal displacement parameters for the protein suggested that the amino terminal of the two domains undergoes 'hinge-bending' motion about an axis passing through the waist of the molecule. Such conformational mobility may be important in allowing access of substrates to the active site of the enzyme. We report here a crystallographic study of a mutant T4 lysozyme which demonstrates further the conformational flexibility of the protein. A mutant form of the enzyme with a methionine residue (Met 6) replaced by isoleucine crystallizes with four independent molecules in the crystal lattice. These four molecules have distinctly different conformations. The mutant protein can also crystallize in standard form with a structure very similar to the wild-type protein. Thus the mutant protein can adopt five different crystal conformations. The isoleucine for methionine substitution at the intersection of the two domains of T4 lysozyme apparently enhances the hinge-bending motion presumed to occur in the wild-type protein, without significantly affecting the catalytic activity or thermal stability of the protein.     

Autonomous forward inference via DNA computing

Recent studies direct the researchers into building DNA computing machines with intelligence, which is measured by three main points: autonomous, programmable and able to learn and adapt. Logical inference plays an important role in programmable information processing or computing. Here we present a new method to perform autonomous molecular forward inference for expert system. A novel repetitive recognition site (RRS) technique is invented to design rule-molecules in knowledge base. The inference engine runs autonomously by digesting the rule-molecule, using a Class IIB restriction enzyme PpiI. Concentration model has been built to show the feasibility of the inference process under ideal chemical reaction conditions. Moreover, we extend to implement a triggering communication between molecular automata, as a further application of the RRS technique in our model.     

Crystal structure of the specificity domain of ribonuclease P

RNase P is the only endonuclease responsible for processing the 5' end of transfer RNA by cleaving a precursor and leading to tRNA maturation. It contains an RNA component and a protein component and has been identified in all organisms. It was one of the first catalytic RNAs identified and the first that acts as a multiple-turnover enzyme in vivo. RNase P and the ribosome are so far the only two ribozymes known to be conserved in all kingdoms of life. The RNA component of bacterial RNase P can catalyse pre-tRNA cleavage in the absence of the RNase P protein in vitro and consists of two domains: a specificity domain and a catalytic domain. Here we report a 3.15-A resolution crystal structure of the 154-nucleotide specificity domain of Bacillus subtilis RNase P. The structure reveals the architecture of this domain, the interactions that maintain the overall fold of the molecule, a large non-helical but well-structured module that is conserved in all RNase P RNA, and the regions that are involved in interactions with the substrate.     

Autonomous forward inference via DNA computing

Recent studies direct the researchers into building DNA computing machines with intelligence, which is measured by three main points: autonomous, programmable and able to learn and adapt. Logical inference plays an important role in programmable information processing or computing. Here we present a new method to perform autonomous molecular forward inference for expert system. A novel repetitive recognition site (RRS) technique is invented to design rule-molecules in knowledge base. The inference engine runs autonomously by digesting the rule-molecule, using a Class IIB restriction enzyme PpiI. Concentration model has been built to show the feasibility of the inference process under ideal chemical reaction conditions. Moreover, we extend to implement a triggering communication between molecular automata, as a further application of the RRS technique in our model.     

Distinct domains of tRNA synthetase recognize the same base pair

Synthesis of proteins containing errors (mistranslation) is prevented by aminoacyl transfer RNA synthetases through their accurate aminoacylation of cognate tRNAs and their ability to correct occasional errors of aminoacylation by editing reactions. A principal source of mistranslation comes from mistaking glycine or serine for alanine, which can lead to serious cell and animal pathologies, including neurodegeneration. A single specific G.U base pair (G3.U70) marks a tRNA for aminoacylation by alanyl-tRNA synthetase. Mistranslation occurs when glycine or serine is joined to the G3.U70-containing tRNAs, and is prevented by the editing activity that clears the mischarged amino acid. Previously it was assumed that the specificity for recognition of tRNA(Ala) for editing was provided by the same structural determinants as used for aminoacylation. Here we show that the editing site of alanyl-tRNA synthetase, as an artificial recombinant fragment, targets mischarged tRNA(Ala) using a structural motif unrelated to that for aminoacylation so that, remarkably, two motifs (one for aminoacylation and one for editing) in the same enzyme independently can provide determinants for tRNA(Ala) recognition. The structural motif for editing is also found naturally in genome-encoded protein fragments that are widely distributed in evolution. These also recognize mischarged tRNA(Ala). Thus, through evolution, three different complexes with the same tRNA can guard against mistaking glycine or serine for alanine.     

Serpin-resistant mutants of human tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) converts the inactive zymogen, plasminogen, into the powerful protease, plasmin, which then degrades the fibrin meshwork of thrombi. To prevent systemic activation of plasminogen, plasma contains several inhibitors of t-PA, the most important of which is plasminogen activator inhibitor-1 (PAI-1), a member of the serpin superfamily. As the ability to produce serpin-resistant variants of t-PA could increase the potential of this enzyme as a thrombolytic agent, we have used the known three-dimensional structure of the complex between trypsin and bovine pancreatic trypsin inhibitor (BPTI) to model the interactions between the active site of human t-PA and PAI-1. On the basis of this model we then altered by site-directed mutagenesis those amino acids of t-PA predicted to make contact with PAI-1 but not with the substrate plasminogen. We report here that although the resulting mutants have enzymatic properties similar to those of wild-type t-PA, they display significant resistance to inhibition by PAI-1. For example, following incubation with an amount of the serpin that completely inhibits the wild-type enzyme, one variant retains 95% of its initial activity. This mutant is also resistant to inhibition by the complex mixture of serpins present in human plasma.     

Structural basis for 5'-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold critical for the endonuclease cleavage activity of RISC. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5'-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5' end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5' end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5' phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere, provide direct support for the 5' region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5' end of the guide RNA.     

Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160.

The envelope glycoprotein of human immunodeficiency virus (HIV) initiates infection by mediating fusion of the viral envelope with the cell membrane. Fusion activity requires proteolytic cleavage of the gp160 protein into gp120 and gp41 at a site containing several arginine and lysine residues. Activation at basic cleavage sites is observed with many membrane proteins of cellular and viral origin. We have recently found that the enzyme activating the haemagglutinin of fowl plague virus (FPV), an avian influenza virus, is furin. Furin, a subtilisin-like eukaryotic endoprotease, has a substrate specificity for the consensus amino-acid sequence Arg-X-Lys/Arg-Arg at the cleavage site. We show here that the glycoprotein of HIV-1, which has the same protease recognition motif as the FPV haemagglutinin, is also activated by furin.     

Role for the nitrogenase MoFe protein alpha-subunit in FeMo-cofactor binding and catalysis

Two components constitute Mo-dependent nitrogenase (EC 1.18.6.1)--the Fe protein (a homodimer encoded by nifH) and the MoFe protein (an alpha 2 beta 2 tetramer encoded by nifDK). The MoFe protein provides the substrate-binding site and probably contains six prosthetic groups of two types--four Fe-S centres and two Fe- and Mo-containing cofactors. To determine the distribution and catalytic function of these metalloclusters, we and others are attempting to change the catalytic and spectroscopic features of nitrogenase by substituting specific amino acids targeted as potential metallocluster ligands, particularly those to the FeMo-cofactor, which is responsible for the biologically unique electron paramagnetic resonance signal (S = 3/2) of nitrogenase, and is believed to be the N2-reducing site. Here we describe mutant strains of Azotobacter vinelandii that have single amino-acid substitutions within the MoFe protein alpha-subunit. These substitutions alter both substrate-reduction properties and the unique electron paramagnetic resonance signal, indicating that the FeMo-cofactor is associated with both the alpha-subunit and the substrate-reducing site.     

Importance of DNA stiffness in protein-DNA binding specificity

From the first high-resolution structure of a repressor bound specifically to its DNA recognition sequence it has been shown that the phage 434 repressor protein binds as a dimer to the helix. Tight, local interactions are made at the ends of the binding site, causing the central four base pairs (bp) to become bent and overtwisted. The centre of the operator is not in contact with protein but repressor binding affinity can be reduced at least 50-fold in response to a sequence change there. This observation might be explained should the structure of the intervening DNA segment vary with its sequence, or if DNA at the centre of the operator resists the torsional and bending deformation necessary for complex formation in a sequence dependent fashion. We have considered the second hypothesis by demonstrating that DNA stiffness is sequence dependent. A method is formulated for calculating the stiffness of any particular DNA sequence, and we show that this predicted relationship between sequence and stiffness can explain the repressor binding data in a quantitative manner. We propose that the elastic properties of DNA may be of general importance to an understanding of protein-DNA binding specificity.