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• W2017232746 abstract "The left-handed parallel β helix (LβH) fold has recently received attention as a possible structure for the prion protein (PrP) in its misfolded state. In light of this interest, we have developed an experimental system to examine the structural requirements of the LβH fold, using a known LβH protein, UDP-N-acetylglucosamine acyltransferase (LpxA), from E. coli. We showed that the β helix can tolerate nonhydrophobic residues at interior positions and prolines were important, but not critical, in folding of the β helix. Using our structural studies of the LβH, we threaded the sequence of the amyloidogenic fragment of the prion protein (residues 104–143) onto the structure of LpxA. Based on the threading result, we constructed the recombinant PrP-LpxA and tested its functional activity in an E. coli antibiotic sensitivity assay. The results of these experiments suggest that the amyloidogenic PrP fragment may fold into a β helix in the context of a larger β-helical structure. The left-handed parallel β helix (LβH) fold has recently received attention as a possible structure for the prion protein (PrP) in its misfolded state. In light of this interest, we have developed an experimental system to examine the structural requirements of the LβH fold, using a known LβH protein, UDP-N-acetylglucosamine acyltransferase (LpxA), from E. coli. We showed that the β helix can tolerate nonhydrophobic residues at interior positions and prolines were important, but not critical, in folding of the β helix. Using our structural studies of the LβH, we threaded the sequence of the amyloidogenic fragment of the prion protein (residues 104–143) onto the structure of LpxA. Based on the threading result, we constructed the recombinant PrP-LpxA and tested its functional activity in an E. coli antibiotic sensitivity assay. The results of these experiments suggest that the amyloidogenic PrP fragment may fold into a β helix in the context of a larger β-helical structure. The parallel β helix is a repetitive protein fold where the repeating unit is a β-helical coil formed by segments of β strands (Choi et al., 2008Choi J.H. Govaerts C. May B.C. Cohen F.E. Analysis of the sequence and structural features of the left-handed beta-helical fold.Proteins. 2008; 73: 150-160Crossref PubMed Scopus (14) Google Scholar, Iengar et al., 2006Iengar P. Joshi N.V. Balaram P. Conformational and sequence signatures in beta helix proteins.Structure. 2006; 14: 529-542Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, Jenkins and Pickersgill, 2001Jenkins J. Pickersgill R. The architecture of parallel beta-helices and related folds.Prog. Biophys. Mol. Biol. 2001; 77: 111-175Crossref PubMed Scopus (196) Google Scholar). Each rung of the canonical β helix consists of two to three β strands interrupted by turn or loop regions (Iengar et al., 2006Iengar P. Joshi N.V. Balaram P. Conformational and sequence signatures in beta helix proteins.Structure. 2006; 14: 529-542Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, Kajava and Steven, 2006Kajava A.V. Steven A.C. Beta-rolls, beta-helices, and other beta-solenoid proteins.Adv. Protein Chem. 2006; 73: 55-96Crossref PubMed Scopus (99) Google Scholar, Simkovsky and King, 2006Simkovsky R. King J. An elongated spine of buried core residues necessary for in vivo folding of the parallel beta-helix of P22 tailspike adhesin.Proc. Natl. Acad. Sci. USA. 2006; 103: 3575-3580Crossref PubMed Scopus (25) Google Scholar). The β-helical rungs are aligned to form a cross-β structure, such that elongated β sheets connected by hydrogen bonds run perpendicular to the helical axis (Choi et al., 2008Choi J.H. Govaerts C. May B.C. Cohen F.E. Analysis of the sequence and structural features of the left-handed beta-helical fold.Proteins. 2008; 73: 150-160Crossref PubMed Scopus (14) Google Scholar, Govaerts et al., 2004Govaerts C. Wille H. Prusiner S.B. Cohen F.E. Evidence for assembly of prions with left-handed beta-helices into trimers.Proc. Natl. Acad. Sci. USA. 2004; 101: 8342-8347Crossref PubMed Scopus (461) Google Scholar, Simkovsky and King, 2006Simkovsky R. King J. An elongated spine of buried core residues necessary for in vivo folding of the parallel beta-helix of P22 tailspike adhesin.Proc. Natl. Acad. Sci. USA. 2006; 103: 3575-3580Crossref PubMed Scopus (25) Google Scholar). The repetition of β helix coils creates a cylindrical hydrophobic core. The hydrophobic core of a β-helical protein is characterized by buried stacks of similar side chains (Choi et al., 2008Choi J.H. Govaerts C. May B.C. Cohen F.E. Analysis of the sequence and structural features of the left-handed beta-helical fold.Proteins. 2008; 73: 150-160Crossref PubMed Scopus (14) Google Scholar, Jenkins and Pickersgill, 2001Jenkins J. Pickersgill R. The architecture of parallel beta-helices and related folds.Prog. Biophys. Mol. Biol. 2001; 77: 111-175Crossref PubMed Scopus (196) Google Scholar, Simkovsky and King, 2006Simkovsky R. King J. An elongated spine of buried core residues necessary for in vivo folding of the parallel beta-helix of P22 tailspike adhesin.Proc. Natl. Acad. Sci. USA. 2006; 103: 3575-3580Crossref PubMed Scopus (25) Google Scholar). While right-handed β helices (RβH) are generally characterized by β strands connected by variable length turns and loops (Heffron et al., 1998Heffron S. Moe G.R. Sieber V. Mengaud J. Cossart P. Vitali J. Jurnak F. Sequence profile of the parallel beta helix in the pectate lyase superfamily.J. Struct. Biol. 1998; 122: 223-235Crossref PubMed Scopus (37) Google Scholar, Iengar et al., 2006Iengar P. Joshi N.V. Balaram P. Conformational and sequence signatures in beta helix proteins.Structure. 2006; 14: 529-542Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, Jenkins and Pickersgill, 2001Jenkins J. Pickersgill R. The architecture of parallel beta-helices and related folds.Prog. Biophys. Mol. Biol. 2001; 77: 111-175Crossref PubMed Scopus (196) Google Scholar), the right-handed β helix (LβH) is more rigid and repetitive than the RβH variant (Choi et al., 2008Choi J.H. Govaerts C. May B.C. Cohen F.E. Analysis of the sequence and structural features of the left-handed beta-helical fold.Proteins. 2008; 73: 150-160Crossref PubMed Scopus (14) Google Scholar, Iengar et al., 2006Iengar P. Joshi N.V. Balaram P. Conformational and sequence signatures in beta helix proteins.Structure. 2006; 14: 529-542Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, Jenkins and Pickersgill, 2001Jenkins J. Pickersgill R. The architecture of parallel beta-helices and related folds.Prog. Biophys. Mol. Biol. 2001; 77: 111-175Crossref PubMed Scopus (196) Google Scholar). It has been proposed that misfolded proteins associated with neurodegenerative diseases, such as prion disease, may adopt an LβH fold (Govaerts et al., 2004Govaerts C. Wille H. Prusiner S.B. Cohen F.E. Evidence for assembly of prions with left-handed beta-helices into trimers.Proc. Natl. Acad. Sci. USA. 2004; 101: 8342-8347Crossref PubMed Scopus (461) Google Scholar, Langedijk et al., 2006Langedijk J.P. Fuentes G. Boshuizen R. Bonvin A.M. Two-rung model of a left-handed beta-helix for prions explains species barrier and strain variation in transmissible spongiform encephalopathies.J. Mol. Biol. 2006; 360: 907-920Crossref PubMed Scopus (49) Google Scholar, Stork et al., 2005Stork M. Giese A. Kretzschmar H.A. Tavan P. Molecular dynamics simulations indicate a possible role of parallel beta-helices in seeded aggregation of poly-Gln.Biophys. J. 2005; 88: 2442-2451Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, Wille et al., 2002Wille H. Michelitsch M.D. Guenebaut V. Supattapone S. Serban A. Cohen F.E. Agard D.A. Prusiner S.B. Structural studies of the scrapie prion protein by electron crystallography.Proc. Natl. Acad. Sci. USA. 2002; 99: 3563-3568Crossref PubMed Scopus (354) Google Scholar, Yang et al., 2005Yang S. Levine H. Onuchic J.N. Cox D.L. Structure of infectious prions: stabilization by domain swapping.FASEB J. 2005; 19: 1778-1782Crossref PubMed Scopus (35) Google Scholar). Recent solid-state nuclear magnetic resonance (NMR) studies of the fungal HET-s prion protein showed that the misfolded amyloid conformation may adopt an architecture that is structurally unrelated to the native conformations, but that is similar to a β helix or β solenoid fold (Kajava and Steven, 2006Kajava A.V. Steven A.C. Beta-rolls, beta-helices, and other beta-solenoid proteins.Adv. Protein Chem. 2006; 73: 55-96Crossref PubMed Scopus (99) Google Scholar, Wasmer et al., 2008Wasmer C. Lange A. Van Melckebeke H. Siemer A.B. Riek R. Meier B.H. Amyloid fibrils of the HET-s(218-289) prion form a beta solenoid with a triangular hydrophobic core.Science. 2008; 319: 1523-1526Crossref PubMed Scopus (779) Google Scholar). Hence, it is plausible that mammalian prion proteins may also adopt β-helical architecture in the misfolded state. Misfolded amyloid proteins share common structural characteristics, even when the native proteins are evolutionarily or structurally unrelated. Amyloid fibrils are generally unbranched, protease-resistant filaments with dominant β sheet structures organized in a cross-β fashion in which the β strands run perpendicular to the fibril axis (Jimenez et al., 2002Jimenez J.L. Nettleton E.J. Bouchard M. Robinson C.V. Dobson C.M. Saibil H.R. The protofilament structure of insulin amyloid fibrils.Proc. Natl. Acad. Sci. USA. 2002; 99: 9196-9201Crossref PubMed Scopus (685) Google Scholar, Murali and Jayakumar, 2005Murali J. Jayakumar R. Spectroscopic studies on native and protofibrillar insulin.J. Struct. Biol. 2005; 150: 180-189Crossref PubMed Scopus (30) Google Scholar, Serpell et al., 2007Serpell L.C. Benson M. Liepnieks J.J. Fraser P.E. Structural analyses of fibrinogen amyloid fibrils.Amyloid. 2007; 14: 199-203Crossref PubMed Scopus (30) Google Scholar). Previous prion amyloid modeling studies have converged on the β-helical architecture due to the structural features that β-helical folds share with the unresolved structure of longer chain amyloids. It has also been observed that expression of the isolated β-helical domain of the RβH protein, P22 tailspike protein, readily forms amyloid-like fibers (Schuler et al., 1999Schuler B. Rachel R. Seckler R. Formation of fibrous aggregates from a non-native intermediate: the isolated P22 tailspike beta-helix domain.J. Biol. Chem. 1999; 274: 18589-18596Crossref PubMed Scopus (36) Google Scholar). Therefore, understanding the role of amino acid sequence in the folding of β helices may provide insight into how misfolded proteins could form elongated β sheet structures. E. coli UDP-N-acetylglucosamine acyltransferase (LpxA) was the first example of a protein where an LβH is the predominant secondary structure (Raetz and Roderick, 1995Raetz C.R. Roderick S.L. A left-handed parallel beta helix in the structure of UDP-N-acetylglucosamine acyltransferase.Science. 1995; 270: 997-1000Crossref PubMed Scopus (283) Google Scholar). LpxA is a soluble, cytoplasmic protein that catalyzes the first step in the biosynthesis of lipid A, the hydrophobic anchor of lipopolysaccharides in gram-negative bacteria (Galloway and Raetz, 1990Galloway S.M. Raetz C.R. A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis.J. Biol. Chem. 1990; 265: 6394-6402Abstract Full Text PDF PubMed Google Scholar, Wyckoff and Raetz, 1999Wyckoff T.J. Raetz C.R. The active site of Escherichia coli UDP-N-acetylglucosamine acyltransferase. Chemical modification and site-directed mutagenesis.J. Biol. Chem. 1999; 274: 27047-27055Crossref PubMed Scopus (65) Google Scholar). Lipid A is required for the growth of E. coli and most other gram-negative bacteria and is also necessary for maintaining the integrity of the outer membrane as a barrier to toxic chemicals (Galloway and Raetz, 1990Galloway S.M. Raetz C.R. A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis.J. Biol. Chem. 1990; 265: 6394-6402Abstract Full Text PDF PubMed Google Scholar, Vaara, 1993Vaara M. Antibiotic-supersusceptible mutants of Escherichia coli and Salmonella typhimurium.Antimicrob. Agents Chemother. 1993; 37: 2255-2260Crossref PubMed Scopus (132) Google Scholar). LpxA monomers are composed of a single β-helical domain, capped at the C terminus with an α-helical domain; they assemble into a homotrimer to form the active enzyme. The β-helical domain of LpxA contains approximately ten rungs (or coils) with two loop excursions (Figure 1). Three repeats of imperfect hexapeptide motifs ([LIV]-[GAED]-X2-[STAV]-X) make up one rung of the β helix (Raetz and Roderick, 1995Raetz C.R. Roderick S.L. A left-handed parallel beta helix in the structure of UDP-N-acetylglucosamine acyltransferase.Science. 1995; 270: 997-1000Crossref PubMed Scopus (283) Google Scholar). Each rung of the canonical β helix consists of three flat and untwisted parallel β strands connected by either a one- or two-residue turn or a long loop excursion region (Choi et al., 2008Choi J.H. Govaerts C. May B.C. Cohen F.E. Analysis of the sequence and structural features of the left-handed beta-helical fold.Proteins. 2008; 73: 150-160Crossref PubMed Scopus (14) Google Scholar, Iengar et al., 2006Iengar P. Joshi N.V. Balaram P. Conformational and sequence signatures in beta helix proteins.Structure. 2006; 14: 529-542Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, Jenkins and Pickersgill, 2001Jenkins J. Pickersgill R. The architecture of parallel beta-helices and related folds.Prog. Biophys. Mol. Biol. 2001; 77: 111-175Crossref PubMed Scopus (196) Google Scholar, Kajava and Steven, 2006Kajava A.V. Steven A.C. Beta-rolls, beta-helices, and other beta-solenoid proteins.Adv. Protein Chem. 2006; 73: 55-96Crossref PubMed Scopus (99) Google Scholar). Previous studies have shown defined residue distributions at the various positions of the repeats: each β strand contains small, uncharged residues (A, S, T, and C) and conserved, larger hydrophobic residues (L, I, and V) that face the interior of the LβH to create a hydrophobic core (Choi et al., 2008Choi J.H. Govaerts C. May B.C. Cohen F.E. Analysis of the sequence and structural features of the left-handed beta-helical fold.Proteins. 2008; 73: 150-160Crossref PubMed Scopus (14) Google Scholar, Iengar et al., 2006Iengar P. Joshi N.V. Balaram P. Conformational and sequence signatures in beta helix proteins.Structure. 2006; 14: 529-542Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, Jenkins and Pickersgill, 2001Jenkins J. Pickersgill R. The architecture of parallel beta-helices and related folds.Prog. Biophys. Mol. Biol. 2001; 77: 111-175Crossref PubMed Scopus (196) Google Scholar, Kajava and Steven, 2006Kajava A.V. Steven A.C. Beta-rolls, beta-helices, and other beta-solenoid proteins.Adv. Protein Chem. 2006; 73: 55-96Crossref PubMed Scopus (99) Google Scholar). These constraints on the interior positions of the β strands are presumed to have restricted sequence variation of the LβH proteins throughout evolution (Parisi and Echave, 2001Parisi G. Echave J. Structural constraints and emergence of sequence patterns in protein evolution.Mol. Biol. Evol. 2001; 18: 750-756Crossref PubMed Scopus (70) Google Scholar). The LβH fold is highly regular and symmetrical with little variability in shape or size over the length of the domain (Zheng et al., 2007Zheng J. Zanuy D. Haspel N. Tsai C.J. Aleman C. Nussinov R. Nanostructure design using protein building blocks enhanced by conformationally constrained synthetic residues.Biochemistry. 2007; 46: 1205-1218Crossref PubMed Scopus (36) Google Scholar). These features also have led to the suggestion that LβH fold may be used as a building block for nanotubular structures with application in nanotechnology (Haspel et al., 2006Haspel N. Zanuy D. Aleman C. Wolfson H. Nussinov R. De novo tubular nanostructure design based on self-assembly of beta-helical protein motifs.Structure. 2006; 14: 1137-1148Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, Haspel et al., 2007Haspel N. Zanuy D. Zheng J. Aleman C. Wolfson H. Nussinov R. Changing the charge distribution of beta-helical-based nanostructures can provide the conditions for charge transfer.Biophys. J. 2007; 93: 245-253Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, Zanuy et al., 2007aZanuy D. Rodriguez-Ropero F. Haspel N. Zheng J. Nussinov R. Aleman C. Stability of tubular structures based on beta-helical proteins: self-assembled versus polymerized nanoconstructs and wild-type versus mutated sequences.Biomacromolecules. 2007; 8: 3135-3146Crossref PubMed Scopus (10) Google Scholar, Zheng et al., 2007Zheng J. Zanuy D. Haspel N. Tsai C.J. Aleman C. Nussinov R. Nanostructure design using protein building blocks enhanced by conformationally constrained synthetic residues.Biochemistry. 2007; 46: 1205-1218Crossref PubMed Scopus (36) Google Scholar). With this interest, there have also been recent studies to improve the stability of the β helix structure using conformationally constrained amino acids (Ballano et al., 2008Ballano G. Zanuy D. Jimenez A.I. Cativiela C. Nussinov R. Aleman C. Structural analysis of a beta-helical protein motif stabilized by targeted replacements with conformationally constrained amino acids.J. Phys. Chem. B. 2008; 112: 13101-13115Crossref PubMed Scopus (15) Google Scholar, Zanuy et al., 2007bZanuy D. Rodriguez-Ropero F. Nussinov R. Aleman C. Testing beta-helix terminal coils stability by targeted substitutions with non-proteogenic amino acids: a molecular dynamics study.J. Struct. Biol. 2007; 160: 177-189Crossref PubMed Scopus (7) Google Scholar). Despite growing interest in the LβH, there has been little structural characterization of the LβH fold. The accuracy of amyloid modeling studies using the LβH fold has been limited by the relative absence of information pertaining to the sequence requirements and structural features of this relatively rare protein fold. In the present study, we investigated the folding and stability of the LpxA protein to examine the structural requirements of the LβH fold by altering the sequence in the β-helical domain. We showed that some nonhydrophobic residues could be tolerated at interior positions of the β helix and that proline residues were important, but not critical, in folding of the β helix. We designed a recombinant PrP-LpxA protein, in which a PrP fragment that is thought to be essential for the conformational conversion was incorporated into the β-helical domain of LpxA. Partial enzymatic activity was observed, suggesting that the β-helical structure may be able to accommodate a portion of the PrP sequence and, as a corollary, that a PrP fragment may adopt LβH architecture. We developed a bioassay to evaluate the structural integrity of LpxA using LpxA enzymatic activity. The E. coli strain SM101 is deficient in LpxA activity due to a G189S inactivating mutation (glycine being the only residue allowed at position 189) in the chromosomal gene (Galloway and Raetz, 1990Galloway S.M. Raetz C.R. A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis.J. Biol. Chem. 1990; 265: 6394-6402Abstract Full Text PDF PubMed Google Scholar, Odegaard et al., 1997Odegaard T.J. Kaltashov I.A. Cotter R.J. Steeghs L. van der Ley P. Khan S. Maskell D.J. Raetz C.R. Shortened hydroxyacyl chains on lipid A of Escherichia coli cells expressing a foreign UDP-N-acetylglucosamine O-acyltransferase.J. Biol. Chem. 1997; 272: 19688-19696Crossref PubMed Scopus (55) Google Scholar). This strain is defective in lipid A biosynthesis and is temperature sensitive, showing no growth at 37°C. At its permissive growth temperature of 30°C, SM101 displays hypersensitivity to antibiotics such as novobiocin, rifampin, and erythromycin that are normally excluded by the outer membrane (Odegaard et al., 1997Odegaard T.J. Kaltashov I.A. Cotter R.J. Steeghs L. van der Ley P. Khan S. Maskell D.J. Raetz C.R. Shortened hydroxyacyl chains on lipid A of Escherichia coli cells expressing a foreign UDP-N-acetylglucosamine O-acyltransferase.J. Biol. Chem. 1997; 272: 19688-19696Crossref PubMed Scopus (55) Google Scholar, Vuorio and Vaara, 1992Vuorio R. Vaara M. The lipid A biosynthesis mutation lpxA2 of Escherichia coli results in drastic antibiotic supersusceptibility.Antimicrob. Agents Chemother. 1992; 36: 826-829Crossref PubMed Scopus (82) Google Scholar). When SM101 is transformed with plasmids containing the wild-type lpxA gene, normal growth is restored (Galloway and Raetz, 1990Galloway S.M. Raetz C.R. A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis.J. Biol. Chem. 1990; 265: 6394-6402Abstract Full Text PDF PubMed Google Scholar). Therefore, enzymatic activity of any LpxA mutant can be directly tested in a transformed SM101 strain exposed to various concentrations of antibiotics. We constructed pJC1 (His tag LpxA) and pJC2 (His tag LpxA mutant containing a single tyrosine) plasmids as described in the Experimental Procedures. pJC1 contained the gene for wild-type LpxA, while pJC2 contained the gene for LpxA, where all tyrosine residues except one were substituted with either phenylalanine or histidine (Y66F, Y77F, Y219F, Y223F, and Y243H). pJC2 had a relatively uncomplicated spectroscopic signature, facilitating the characterization of the folded and unfolded states. Negative control mutants were made by site-directed mutagenesis at H125A and I86R of lpxA to generate mutant pJC2-H125A and pJC2-I86R, respectively. H125 is an important residue in the LpxA active site and its mutation almost completely eliminates LpxA activity (Williams and Raetz, 2007Williams A.H. Raetz C.R. Structural basis for the acyl chain selectivity and mechanism of UDP-N-acetylglucosamine acyltransferase.Proc. Natl. Acad. Sci. USA. 2007; 104: 13543-13550Crossref PubMed Scopus (53) Google Scholar, Wyckoff and Raetz, 1999Wyckoff T.J. Raetz C.R. The active site of Escherichia coli UDP-N-acetylglucosamine acyltransferase. Chemical modification and site-directed mutagenesis.J. Biol. Chem. 1999; 274: 27047-27055Crossref PubMed Scopus (65) Google Scholar). The I86 residue is located in the fifth rung of the β-helical domain in the hydrophobic core of a β helix (Figure 1A). Based on our examination of the structure, a substitution at I86 with a large, charged residue, such as arginine, was targeted to promote improper folding or destabilization of the β-helical domain and thereby decrease LpxA activity of the pJC2-I86R mutant. Hence, H125A (pJC2-H125A) was used as a functional negative control mutant and I86R (pJC2-I86R) was used as a structural negative control mutant. Both mutants were used to test whether an LpxA functional assay could be used as an in vivo folding assay for β-helical folding. The SM101 (DE3) strain was transformed with pJC1, pJC2, pJC2-H125A, and pJC2-I86R plasmids. Selected colonies of each transformant and SM105 (isogenic wild-type strain of SM101) were grown overnight, then transferred to 96 well plates and grown in medium dosed ampicillin (50 μg/ml), streptomycin (30 μg/ml), and novobiocin (0–256 μg/ml) at 30°C. Novobiocin was used to determine the antibiotic susceptibility of the SM101 (DE3) strain once transformed with either wild-type LpxA or the mutant plasmids. Bacterial growth was quantified after 12 hr by measuring the optical density at 600 nm (OD600) and expressed as a function of novobiocin concentration. Introduction of the wild-type lpxA gene allowed E. coli containing the lpxA2 mutation to grow under the same range of novobiocin concentrations as SM105. As shown in Figure 2, the single tyrosine mutant construct, pJC2, also showed growth similar to wild-type E. coli. Therefore, pJC2 was used as a wild-type LpxA control in the present study. In contrast, pJC2-H125A and pJC2-I86R showed significant reductions in growth and hypersensitivity to novobiocin (minimum inhibitory concentration of 4 μg/ml versus 256 μg/ml for wild-type). This result was consistent with previous studies of SM101 (Vuorio and Vaara, 1992Vuorio R. Vaara M. The lipid A biosynthesis mutation lpxA2 of Escherichia coli results in drastic antibiotic supersusceptibility.Antimicrob. Agents Chemother. 1992; 36: 826-829Crossref PubMed Scopus (82) Google Scholar). Note that no isopropyl β-D-1-thiogalatopyranoside (IPTG) induction was required, as the basal protein expression was sufficient to recover LpxA activity. Protein expression was verified by western blot using an anti-his antibody (data not shown). We first aimed at identifying the best site to probe structural requirements of the LβH in LpxA, i.e., where structural perturbations will lead to a functional phenotype. Therefore we conducted an arginine scan of the β-helical domain by site-directed mutagenesis at hydrophobic residues in the β-helical core, located in rungs 1–7 (Figure 1A). An in vivo folding assay with novobiocin was conducted at 30°C with the LpxA mutants I2R, I20R, I38R, I56R, I86R, V111R, and V129R along with controls, as described earlier (Figure 3A). The log LD50 (50% of the lethal dose concentration) was calculated from the bacterial growth curve plotted against the novobiocin concentration. Log LD50 was then plotted against the location of mutagenesis sites, referenced by rung number (Figure 3B). The arginine scan of the hydrophobic core of the β-helical domain showed that LpxA can be fully or partially active, even with the disruption of up to three rungs (I38R) of the β-helical domain. However, an arginine mutation at I56 eliminated the activity of LpxA. Therefore, based on our arginine scan and structural analysis of the active site, we identified a region of rung 4 (residues 51–62) as an ideal target for studying the folding and stability of the β-helical region. In order to study the effect of nonhydrophobic residues in the core of the β-helical region on LpxA folding, we conducted site-directed mutagenesis at residue I56, substituting with alanine, asparagine, glutamine, glycine, and arginine. SM101 (DE3) was transformed with the LpxA mutants and the in vivo folding assay was performed at 30°C. The assay was also conducted at 37°C in order to test whether SM101 (DE3) transformed with LpxA mutants could restore E. coli growth and to compare the effect of mutations on LpxA folding at a normal growth temperature. Wild-type LpxA and negative control (LpxA-H125A) mutants were also characterized in the in vivo folding assay. At 30°C, substitution of I56 with alanine did not significantly affect the in vivo LpxA activity, but glycine, asparagine, and glutamine mutations showed lower LpxA activity than the wild-type. The arginine mutant was completely inactive (Figure 4A). At 37°C, wild-type, alanine, asparagine, glutamine, and glycine mutations showed similar levels of LpxA activity within the range of standard deviation, but their LpxA activities at 37°C were decreased by 2- to 4-fold compared to those at 30°C. The arginine mutants showed complete loss of LpxA activity at 37°C (Figure 4B). To assess these results at the level of protein expression, LpxA mutants were individually overexpressed using IPTG induction at 30°C. Soluble and insoluble fractions of the lysates were separated by centrifugation and the samples were analyzed by electrophoresis on an SDS polyacrylamide gel. As can be seen in Figure 4C, wild-type LpxA, LpxA-H125A, and the alanine and asparagine LpxA mutants were found mostly in the soluble fractions. Weaker LpxA bands were observed from the soluble fractions of lysates from the glutamine mutant bacteria. LpxA from the glycine and arginine mutant bacteria were found mostly in the insoluble fraction, indicating that the arginine mutation had a detrimental effect on folding of the β helix. Although the expression level of soluble glycine LpxA mutant was lower than expected from the LpxA in vivo folding assay, protein expression of the LpxA mutants was consistent overall with the LpxA in vivo folding assay. Surveys of known LβH structures indicate that proline residues are restricted to defined positions, suggesting a possible role in folding and stability (Choi et al., 2008Choi J.H. Govaerts C. May B.C. Cohen F.E. Analysis of the sequence and structural features of the left-handed beta-helical fold.Proteins. 2008; 73: 150-160Crossref PubMed Scopus (14) Google Scholar). To investigate any possible role for prolines in folding and stability, two proline residues (P28 and P34) and four proline residues (P10, P28, P34, and P183) at the T1 positions of the β-helical domain were replaced with alanine using site-directed mutagenesis. These were termed PtoA-2 and PtoA-4, respectively. The P182 residue, which is located in the unusual turn position of Bo4, was excluded from our scanning, due to its deleterious effect on LpxA folding when mutated to glycine or alanine (data not shown). Results from the in vivo folding assay showed that the activity of the PtoA-4 mutant was lower than wild-type LpxA, but still greater than the negative control, LpxA-H125A. The PtoA-2 mutant showed greater activity than the PtoA-4 mutant (Figure 5A). The thermodynamic stability of the wild-type and proline LpxA mutant was probed by tyrosine fluorescence spectroscopy. We used a modified LpxA background where all but a single tyrosine residue (Y184) were replaced with phenylalanine or histidine in order to simplify the fluorescence spectrum during folding and unfolding. As Y184 is protected from the solvent by the C-terminal domain, unfolding of the protein will lead to increased exposure of Y184 that will translate into increased fluorescence intensity. The mutant proteins were expressed and purified, as described in the Experimental Procedures section. Upon excitation at 280 nm, maximal fluorescent emission was observed at 303 nm (Figur" @default.
• W2017232746 created "2016-06-24" @default.
• W2017232746 creator A5009424761 @default.
• W2017232746 creator A5044762631 @default.
• W2017232746 creator A5052367502 @default.
• W2017232746 creator A5086726231 @default.
• W2017232746 date "2009-07-01" @default.
• W2017232746 modified "2023-09-27" @default.
• W2017232746 title "Site-Directed Mutagenesis Demonstrates the Plasticity of the β Helix: Implications for the Structure of the Misfolded Prion Protein" @default.
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