From monomeric to homodimeric endonucleases and back: engineering novel specificity of LAGLIDADG enzymes

Monomeric homing endonucleases of the LAGLIDADG family recognize DNA in a bipartite manner, reflecting the underlying structural assembly of two protein domains (A and B) related by pseudo 2-fold symmetry. This architecture allows for changes in DNA specificity via the distinct combination of these half-site domains. The key to engineering such hybrid proteins lies in the LAGLIDADG two-helix bundle that forms both the domain interface and the endonuclease active site. In this study, we utilize domain A of the monomeric I-DmoI to demonstrate the feasibility of generating functional homodimeric endonucleases that recognize palindromic DNA sequences derived from the original, non-palindromic target. Wild-type I-DmoI domain A is capable of forming a homodimer (H-DmoA) that binds tightly to, but does not cleave efficiently, its anticipated DNA target. Partial restoration of DNA cleavage ability was obtained by re-engineering the LAGLIDADG dimerization interface (H-DmoC). Upon fusing two copies of H-DmoC via a short peptide linker, a novel, site-specific DNA endonuclease was created (H-DmoC2). Like I-DmoI, H-DmoC2 is thermostable and cleaves the new target DNA to generate the predicted 4 nt 3'-OH overhangs but, unlike I-DmoI, H-DmoC2 retains stringent cleavage specificity when substituting Mn2+ for Mg2+ as co-factor. This novel endonuclease allows speculation regarding specificity of monomeric LAGLIDADG proteins, while it supports the evolutionary genesis of these proteins by a gene duplication event.     

Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space

LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ≈ 20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering-many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.     

Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases

LAGLIDADG homing endonucleases (LHEs) cleave 18–24bp DNA sequences and are promising enzymes for applications requiring sequence-specific DNA cleavage amongst genome-sized DNA backgrounds. Here, we report a method for cell surface display of LHEs, which facilitates analysis of their DNA binding and cleavage properties by flow cytometry. Cells expressing surface LHEs can be stained with fluorescently conjugated double-stranded oligonucleotides (dsOligos) containing their respective target sequences. The signal is absolutely sequence specific and undetectable with dsOligos carrying single base-pair substitutions. LHE–dsOligo interactions facilitate rapid enrichment and viable recovery of rare LHE expressing cells by both fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS). Additionally, dsOligos conjugated with unique fluorophores at opposite termini can be tethered to the cell surface and used to detect DNA cleavage. Recapitulation of DNA binding and cleavage by surface-displayed LHEs provides a high-throughput approach to library screening that should facilitate rapid identification and analysis of enzymes with novel sequence specificities.     

An analysis of protein domain linkers: their classification and role in protein folding

Recent advances in protein engineering have come from creating multi-functional chimeric proteins containing modules from various proteins. These modules are typically joined via an oligopeptide linker, the correct design of which is crucial for the desired function of the chimeric protein. Here we analyse the properties of naturally occurring inter-domain linkers with the aim to design linkers for domain fusion. Two main types of linker were identified; helical and non-helical. Helical linkers are thought to act as rigid spacers separating two domains. Non-helical linkers are rich in prolines, which also leads to structural rigidity and isolation of the linker from the attached domains. This means that both linker types are likely to act as a scaffold to prevent unfavourable interactions between folding domains. Based on these results we have constructed a linker database intended for the rational design of linkers for domain fusion, which can be accessed via the Internet at http://mathbio.nimr.mrc.ac.uk.     

新型四价抗体的优势与基因工程制备方法

近年来基因工程抗体技术发展迅速,将单价小分子抗体改建为双价或多价,成为抗体工程中活跃的领域之一.四价抗体相比于单价或双价抗体在增加抗体亲和力、降低解离率、延长半寿期等药代动力学,受体交联效应等方面具有优势.因此,具有较好的研究和临床应用前景.制备四价抗体的方法主要有化学偶联法和基因工程法,后者主要包括：①通过改变单链抗体VH和VL之间的连接肽的长短对单链抗体之间的聚合作用产生影响;②利用连接肽将scFv进行连接而形成多价抗体分子;③通过寡聚化的结构域,如亮氨酸拉链结构、链亲和素、人p53四聚化功能域、子宫球蛋白等介导多价抗体的形成.     

High-resolution crystal structure of spectrin SH3 domain fused with a proline-rich peptide

A new chimeric protein, named WT-CIIA, was designed by connecting the proline-rich decapeptide PPPVPPYSAG to the C-terminus of the alpha-spectrin SH3 domain through a natural twelve-residue linker to obtain a single-chain model that would imitate intramolecular SH3-ligand interaction. The crystal structure of this fusion protein was determined at 1.7 ? resolution. The asymmetric unit of the crystal contained two SH3 globules contacting with one PPPVPPY fragment located between them. The domains are related by the two-fold non-crystallographic axis and the ligand lies in two opposite orientations with respect to the conservative binding sites of SH3 domains.     

Mapping of a DNA binding region of the PI-sceI homing endonuclease by affinity cleavage and alanine-scanning mutagenesis.

The PI-SceI protein is a member of the LAGLIDADG family of homing endonucleases that is generated by a protein splicing reaction. PI-SceI has a bipartite domain structure, and the protein splicing and endonucleolytic reactions are catalyzed by residues in domains I and II, respectively. Structural and mutational evidence indicates that both domains mediate DNA binding. Treatment of the protein with trypsin breaks a peptide bond within a disordered region of the endonuclease domain situated between residues Val-270 and Leu-280 and interferes with the ability of this domain to bind DNA. To identify specific residues in this region that are involved in DNA binding and/or catalysis, alanine-scanning mutagenesis was used to create a set of PI-SceI mutant proteins that were assayed for activity. One of these mutants, N281A, was >300-fold less active than wild-type PI-SceI, and two other proteins, R277A and N284A, were completely inactive. These decreases in cleavage activity parallel similar decreases in substrate binding by the endonuclease domains of these mutant proteins. We mapped the approximate position of the disordered region to one of the ends of the 31 base pair PI-SceI recognition sequence using mutant proteins that were substituted with cysteine at residues Asn-274 and Glu-283 and tethered to the chemical nuclease FeBABE. These mutational and affinity cleavage data strongly support a model of PI-SceI docked to its DNA substrate that suggests that one or more residues identified here are responsible for contacting base pair A/T(-)(9), which is essential for substrate binding.     

Unusual evolutionary history of the tRNA splicing endonuclease EndA: relationship to the LAGLIDADG and PD-(D/E)XK deoxyribonucleases

The tRNA splicing endoribonuclease EndA from Methanococcus jannaschii is a homotetramer formed via heterologous interaction between the two pairs of homodimers. Each monomer consists of two alpha/beta domains, the N-terminal domain (NTD) and the C-terminal domain (CTD) containing the RNase A-like active site. Comparison of the EndA coordinates with the publicly available protein structure database revealed the similarity of both domains to site-specific deoxyribonucleases: the NTD to the LAGLIDADG family and the CTD to the PD-(D/E)XK family. Superposition of the NTD on the catalytic domain of LAGLIDADG homing endonucleases allowed a suggestion to be made about which amino acid residues of the tRNA splicing nuclease might participate in formation of a presumptive cryptic deoxyribonuclease active site. On the other hand, the CTD and PD-(D/E)XK endonucleases, represented by restriction enzymes and a phage lambda exonuclease, were shown to share extensive similarities of the structural framework, to which entirely different active sites might be attached in two alternative locations. These findings suggest that EndA evolved from a fusion protein with at least two distinct endonuclease activities: the ribonuclease, which made it an essential "antitoxin" for the cells whose RNA genes were interrupted by introns, and the deoxyribonuclease, which provided the means for homing-like mobility. The residues of the noncatalytic CTDs from the positions corresponding to the catalytic side chains in PD-(D/E)XK deoxyribonucleases map to the surface at the opposite side to the tRNA binding site, for which no function has been implicated. Many restriction enzymes from the PD-(D/E)XK superfamily might have the potential to maintain an additional active or binding site at the face opposite the deoxyribonuclease active site, a property that can be utilized in protein engineering.     

Highly efficient expression and purification system of small-size protein domains in Escherichia coli for biochemical characterization

It is often essential to focus the study on the small-size domains of large proteins in eukaryotic cells in the post-genomic era, but the low expression level, insolubility, and instability of the domains have been continuing to hinder the massive purification of domain peptides for structural and biological investigation. In this work, a highly efficient expression and purification system based on a small-size fusion partner GB1 and histidine tag was utilized to solve these problems. Two vectors, namely pGBTNH and pGBH, were constructed to improve expression and facilitate purification. The linker and thrombin cleavage site have been optimized for minimal degradation during purification process. This system has been tested for eight domain peptides varying in size, linker, hydrophobicity, and predicted secondary structure. The results indicate that this system is achievable to produce these domain peptides with high solubility and stability for further biochemical characterization. Moreover, the fusion protein without the linker and thrombin cleavage site is also suitable for spectroscopic studies especially for NMR structural elucidation, if the target peptide is prone to precipitation or easily degraded during purification. This system will be beneficial to the research field of structure and function of small domain and peptide fragment.     

High-Resolution Crystal Structure of Spectrin SH3 Domain Fused with a Proline-Rich Peptide

Abstract

A new chimeric protein, named WT-CIIA, was designed by connecting the proline-rich decapeptide PPPVPPYSAG to the C-terminus of the alpha-spectrin SH3 domain through a natural twelve-residue linker to obtain a single-chain model that would imitate intramolecular SH3-ligand interaction. The crystal structure of this fusion protein was determined at 1.7 Å resolution. The asymmetric unit of the crystal contained two SH3 globules contacting with one PPPVPPY fragment located between them. The domains are related by the twofold non-crystallographic axis and the ligand lies in two opposite orientations with respect to the conservative binding sites of SH3 domains.     

Stimulation of homologous recombination through targeted cleavage by chimeric nucleases

Chimeric nucleases that are hybrids between a nonspecific DNA cleavage domain and a zinc finger DNA recognition domain were tested for their ability to find and cleave their target sites in living cells. Both engineered DNA substrates and the nucleases were injected into Xenopus laevis oocyte nuclei, in which DNA cleavage and subsequent homologous recombination were observed. Specific cleavage required two inverted copies of the zinc finger recognition site in close proximity, reflecting the need for dimerization of the cleavage domain. Cleaved DNA molecules were activated for homologous recombination; in optimum conditions, essentially 100% of the substrate recombined, even though the DNA was assembled into chromatin. The original nuclease has an 18-amino-acid linker between the zinc finger and cleavage domains, and this enzyme cleaved in oocytes at paired sites separated by spacers in the range of 6 to 18 bp, with a rather sharp optimum at 8 bp. By shortening the linker, we found that the range of effective site separations could be narrowed significantly. With no intentional linker between the binding and cleavage domains, only binding sites exactly 6 bp apart supported efficient cleavage in oocytes. We also showed that two chimeric enzymes with different binding specificities could collaborate to stimulate recombination when their individual sites were appropriately placed. Because the recognition specificity of zinc fingers can be altered experimentally, this approach holds great promise for inducing targeted recombination in a variety of organisms.     

Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) are multimodular proteins capable of producing important peptide natural products. Using an assembly line process, the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation‐PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C‐terminal subdomain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. Proteins 2014; 82:2691–2702. © 2014 Wiley Periodicals, Inc.     

Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study

Background

Human blood coagulation factor VIII (fVIII) is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3) that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa) which serves as a critical co-factor for factor IXa (fIXa) enzyme in the intrinsic coagulation pathway.

Results

In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 Å X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740) and A3-C1-C2 (Ser1669-Tyr2332) domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein.

Conclusions

MD simulations predicted that the acidic linker peptide (a1) between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569) region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1) undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the large body of experimental data that implicated the critical importance of the cleavage of the peptide bond between the Arg372 and Ser373 residues for the full co-factor activity of fVIII.     

Postbinding events mediated by human immunodeficiency virus type 1 are sensitive to modifications in the D4-transmembrane linker region of CD4.

Evidence from both structural and functional studies of the CD4 molecule suggests that several domains, including the transmembrane (TM) domain and the adjoining extracellular region (D4-TM linker), contribute to the post-gp12O-binding events leading to human immunodeficiency virus-mediated membrane fusion. To investigate such a role in syncytium formation and cell-free infectivity, we generated several deletion and substitution mutations in the TM and D4-TM linker regions of the CD4 molecule. We found that while the TM domain of CD4 was dispensable for cell-cell and virus-cell interactions, modifications in the D4-TM linker led to perturbations in both processes. Deletion of the five amino acid residues linking D4 to the TM domain resulted in a delayed and reduced capacity to form syncytia, whereas replacement of the residues with the heterologous sequence from the CD8 molecule restored the kinetic profile to wild-type CD4 levels. On the other hand, both mutants of the CD4 D4-TM linker demonstrated delayed cell-free human immunodeficiency virus type 1 infectivity profiles. The defective fusion capacity may be linked to structural perturbations identified with anti-CD4 monoclonal antibodies in the D1-D2 interface and D3 domain of the deletion mutant yet absent in D1 and D4. While all cells were found to bind comparable levels of gp120, both D4-TM linker mutants appeared to induce a decrease in the V3 loop exposure of bound gp120. This underexposure may explain the delays in cell-free infectivities observed for both of these mutants. Together, these findings confirm a role for regions of the CD4 molecule located outside D1 in post-gp120-binding events and suggest that the D4-TM interface contributes to the conformational changes that direct the fusion process.     

Analysis of the LAGLIDADG interface of the monomeric homing endonuclease I-DmoI

The general structural fold of the LAGLIDADG endonuclease family consists of two similar α/β domains (αββαββα) that assemble either as homodimers or monomers with the domains related by pseudo-two-fold symmetry. At the center of this symmetry is the closely packed LAGLIDADG two-helix bundle that forms the main inter- or intra-molecular contact region between the domains of single- or double-motif proteins, respectively. In this work, we further examine the role of the LAGLIDADG residues involved in the helix–helix interaction. The interchangeability of the LAGLIDADG helix interaction was explored by grafting interfacial residues from the homodimeric I-CreI into the corresponding positions in the monomeric I-DmoI. The resulting LAGLIDADG exchange mutant is partially active, preferring to nick dsDNA rather than making the customary double-strand break. A series of partial revertants within the mutated LAGLIDADG region are shown to restore cleavage activity to varying degrees resulting in one I-DmoI mutant that is more active than wild-type I-DmoI. The phenotype of some of these mutants was reconciled on the basis of similarity to the GxxxG helix interaction found in transmembrane proteins. Additionally, a split variant of I-DmoI was created, demonstrating that the LAGLIDADG helices of I-DmoI are capable of forming and maintaining the protein–protein interface in trans to create an active heterodimer.     

High-level expression of the simian virus 40 genes LP1, VP1 and VP2 as fusion proteins in Escherichia coli

The complete sequences of the SV40 agnogene (LP1) and the genes coding for the capsid proteins VP1 and VP2 have been cloned into Escherichia coli expression plasmids. High levels of expression were obtained when the SV40 genes were inserted into the coding sequence of the influenza virus NS1 gene, which has previously been expressed in E. coli. The NS1A-LP1 and NS1A-VP2 chimeric proteins consist of the 81 N-terminal residues of NS1 (designated as peptide NS1A) fused to the complete sequence of the corresponding SV40 protein. The NS1A-VP1 chimera consists of NS1A followed by a linker of nine arbitrary residues and the complete sequence of the SV40 major capsid protein. The observed levels of expression vary considerably among the three chimeric proteins, ranging from approx. 70 micrograms/ml in the case of NS1A-LP1 to approx. 5 micrograms/ml in the case of NS1A-VP2. Cyanogen bromide cleavage of the NS1A-LP1 fusion protein produces fragments with Mrs expected for isolated NS1A and LP1 peptides. A plasmid has also been constructed which expresses the NS1A peptide in high yield.     

Crystal structure of the thermostable archaeal intron-encoded endonuclease I-DmoI

I-DmoI is a 22 kDa endonuclease encoded by an intron in the 23 S rRNA gene of the hyperthermophilic archaeon Desulfurococcus mobilis. The structure of I-DmoI has been determined to 2.2 A resolution using multi-wavelength anomalous diffraction techniques. I-DmoI, a protein of the LAGLIDADG motif family, represents the first structure of a freestanding endonuclease with two LAGLIDADG motifs, and the first of a thermostable homing endonuclease. I-DmoI consists of two similar alpha/beta domains (alphabetabetaalphabetabetaalpha) related by pseudo 2-fold symmetry. The LAGLIDADG motifs are located at the carboxy-terminal end of the first alpha-helix of each domain. These helices form a two-helix bundle at the interface between the domains and are perpendicular to a saddle-shaped DNA binding surface, formed by two four-stranded antiparallel beta-sheets. Despite substantially different sequences, the overall fold of I-DmoI is similar to that of two other LAGLIDADG proteins for which the structures are known, I-CreI and the endonuclease domain of PI-SceI. The three structures differ most in the loops connecting the beta-strands, relating to the respective DNA target site sizes and geometries. In addition, the absence of conserved residues surrounding the active site, other than those within the LAGLIDADG motif, is of mechanistic importance. Finally, the carboxy-terminal domain of I-DmoI is smaller and has a more irregular fold than the amino-terminal domain, which is more similar to I-CreI, a symmetric homodimeric endonuclease. This is reversed compared to PI-SceI, where the amino-terminal domain is more similar to carboxy-terminal domain of I-DmoI and to I-CreI, with interesting evolutionary implications.     

Length requirements for membrane fusion of influenza virus hemagglutinin peptide linkers to transmembrane or fusion peptide domains

During membrane fusion, the influenza A virus hemagglutinin (HA) adopts an extended helical structure that contains the viral transmembrane and fusion peptide domains at the same end of the molecule. The peptide segments that link the end of this rod-like structure to the membrane-associating domains are approximately 10 amino acids in each case, and their structure at the pH of fusion is currently unknown. Here, we examine mutant HAs and influenza viruses containing such HAs to determine whether these peptide linkers are subject to specific length requirements for the proper folding of native HA and for membrane fusion function. Using pairwise deletions and insertions, we show that the region flanking the fusion peptide appears to be important for the folding of the native HA structure but that mutant proteins with small insertions can be expressed on the cell surface and are functional for membrane fusion. HA mutants with deletions of up to 10 residues and insertions of as many as 12 amino acids were generated for the peptide linker to the viral transmembrane domain, and all folded properly and were expressed on the cell surface. For these mutants, it was possible to designate length restrictions for efficient membrane fusion, as functional activity was observed only for mutants containing linkers with insertions or deletions of eight residues or less. The linker peptide mutants are discussed with respect to requirements for the folding of native HAs and length restrictions for membrane fusion activity.     

Protein footprinting approach to mapping DNA binding sites of two archaeal homing enzymes: evidence for a two-domain protein structure.

The archaeal intron-encoded homing enzymes I-PorI and I-DmoI belong to a family of endonucleases that contain two copies of a characteristic LAGLIDADG motif. These endonucleases cleave their intron- or intein- alleles site-specifically, and thereby facilitate homing of the introns or inteins which encode them. The protein structure and the mechanism of DNA recognition of these homing enzymes is largely unknown. Therefore, we examined these properties of I-PorI and I-DmoI by protein footprinting. Both proteins were susceptible to proteolytic cleavage within regions that are equidistant from each of the two LAGLIDADG motifs. When complexed with their DNA substrates, a characteristic subset of the exposed sites, located in regions immediately after and 40-60 amino acids after each of the LAGLIDADG motifs, were protected. Our data suggest that the enzymes are structured into two, tandemly repeated, domains, each containing both the LAGLIDADG motif and two putative DNA binding regions. The latter contains a potentially novel DNA binding motif conserved in archaeal homing enzymes. The results are consistent with a model where the LAGLIDADG endonucleases bind to their non-palindromic substrates as monomeric enzymes, with each of the two domains recognizing one half of the DNA substrate.