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Distinct structural elements govern folding, stability and catalysis in the outer membrane enzyme PagP Bharat Ramasubramanian Iyer, and Radhakrishnan Mahalakshmi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00678 • Publication Date (Web): 15 Aug 2016 Downloaded from http://pubs.acs.org on August 17, 2016

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Distinct structural elements govern folding, stability and catalysis in the outer membrane enzyme PagP Bharat Ramasubramanian Iyer and Radhakrishnan Mahalakshmi1

Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal – 462066. India.

1

To whom correspondence should be addressed: Dr. R. Mahalakshmi, Ph.D., Associate Professor

and Wellcome Trust/DBT India Alliance Intermediate Fellow, Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Academic Building III, Room # 324, Bhopal Bypass Road, Bhauri, Bhopal - 462066. India. Ph: +91-755-6692562. Fax: +91-755-6692392. E-mail: [email protected].

1 ACS Paragon Plus Environment

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Abbreviations: Cm, chemical denaturation mid-point; CD, circular dichroism; DLPC, 12:0 PC, 1,2-dilauroyl-sn-glycero-3-phosphocholine;

DMPC, 14:0 PC, 1,2-dimyristoyl-sn-glycero-3-

phosphocholine; DPC, n-dodecyl phosphocholine; DPPC, 16:0 PC, 1,2-dipalmitoyl-sn-glycero3-phosphocholine; DSPC, 18:0 PC, 1,2-distearoyl-sn-glycero-3-phosphocholine; fU, unfolded fraction; ΔGU, Gibbs free energy of unfolding; GdnHCl, guanidine hydrochloride; KM, Michaelis constant; LPR, lipid-to-protein ratio; m, unfolding cooperativity; ME215, molar ellipticity at 215 nm; ME231, molar ellipticity at 231 nm; OMP, outer membrane protein; PagP, PhoPQ-activated gene P; PK, proteinase K; Tm, thermal denaturation mid-point; Tm-start, start temperature of thermal denaturation; TLE, E. coli total lipid extract; Vmax, maximum reaction velocity; WT, wild-type.


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ABSTRACT The outer membrane enzyme PagP is indispensable for lipid A palmitoylation in Gramnegative bacteria and has been implicated in resistance to host immune defenses. PagP possesses an unusual structure for an integral membrane protein, with a highly dynamic barrel domain that is tilted with respect to the membrane normal. Further, it contains an N-terminal amphipathic helix. Recent functional and structural studies have shown that these molecular factors are critical for PagP to carry out its function in the challenging environment of the bacterial outer membrane. However, the precise contributions of the N-helix on folding and stability, and residues that can influence catalytic rates remain to be addressed. Here, we identify a sequencedependent stabilizing role for the N-terminal helix of PagP in the measured thermodynamic stability of the barrel. Using chimeric barrel sequences, we show that the Escherichia coli PagP N-terminal helix confers 2-fold greater stability to the Salmonella typhimurium barrel. Further, we find that the substitution W78F in S. typhimrium causes a nearly 20-fold increase in the specific activity in vitro for the phospholipase reaction, compared to E. coli PagP. Here, phenylalanine serves as a key regulator of catalysis, possibly by increasing the reaction rate. Through coevolution analysis, we detect an interaction network between seemingly unrelated segments of this membrane protein. Exchanging the structural and functional features between homologous PagP enzymes from E. coli and S. typhimurium has provided us with an understanding of the molecular factors governing PagP stability and function.

Keywords: membrane enzyme, structure-function, protein evolution, protein stability, enzyme kinetics, biophysics.


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The PhoP-PhoQ virulence system is a vital signaling network that dictates lipid remodeling in the outer membrane of Gram-negative bacteria. One of the PhoPQ-activated proteins – PagP – has been implicated in bacterial resistance to host immune defenses in several organisms such as Salmonella, Legionella, and Bordetella.1-8 PagP, or PhoPQ-activated gene P, is an enzyme found in the outer membrane of several Gram-negative bacteria. It catalyzes the transfer of a palmitoyl chain from glycerophospholipids accumulated in the outer leaflet, to the lipid A component of lipopolysaccharides.7, 9-11 The molecular factors governing the folding, function and regulation of this enzyme have been the subject of detailed investigation. Structural and thermodynamic analyses of Escherichia coli PagP demonstrate that this protein possesses an unusually tilted, lipid solvated yet highly stable β-barrel structure.3, 8, 12-16 The N-terminal amphipathic helix, another unusual occurrence for β-barrel membrane proteins, has been annotated as a post-assembly clamp.14 Although PagP constructs lacking the N-terminal helix can fold, the stability is significantly reduced.14 Using ϕvalue analysis, a pioneering study elucidated the folding pathway of PagP in phosphocholine vesicles.17 This study also showed that the N-terminal helix anchors the barrel, through interactions established late in the folding pathway. However, the molecular mechanism through which this anchoring is brought about remains unanswered. Similarly, the catalytic properties of PagP have been thoroughly investigated. It is known that PagP remains active when the Nterminal helix is deleted.14, 15, 18 The active site residues and their relative orientation, substrate entry and product exit sites have also been identified.19 However, in light of recent findings,10, 20 it is evident that the catalytic mechanism is not yet thoroughly understood. One of the main reasons for this conundrum is the highly dynamic structure of PagP, which makes site-specific structural analysis difficult.21 Substrate binding is expected to cause a


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significant change in the active site region, which is located in the unstructured extracellular loops. Therefore, the available crystal12, 16 and NMR3 structures provide incomplete information on the active site residues. Moreover, an explicit reason behind PagP from different sources showing varied levels of palmitoylation efficiencies is not known.7, 22 In this study, we address these questions using two highly homologous versions of PagP from E. coli and S. typhimurium, namely PagP-Ec and PagP-St, to understand the biophysical and biochemical features of this enzyme. These two proteins share >80% sequence identity, and were anticipated to display similar functional and thermodynamic properties. Surprisingly, our previous study revealed that the PagP homologs exhibit significant differences in the folding, stability, and function.20 Specifically, our findings suggested that although PagP-Ec is two-fold more stable than PagP-St, this stability is offset by the 20-fold higher catalytic efficiency of PagP-St. Through the current study, we account for the differences in PagP, by deciphering the amino acid residues responsible for such remarkable changes in both the β-barrel fold and function. Our deductions can have significant implications in the understanding of “customized” organism-dependent variation of protein stability and function.


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EXPERIMENTAL PROCEDURES Cloning, mutant generation and protein preparation The two wild-type proteins, PagP-Ec (Uniprot ID: P37001) and PagP-St (Uniprot ID: Q8ZR06), consist of 161 residues in the mature form. The N-terminal signal sequence of residue length 25 and 29, respectively, were removed for cytoplasmic expression in E. coli BL21 (DE3) cells as inclusion bodies. The gene for PagP, without the signal sequence, was amplified from the genome of E. coli K-12 MG1655 and S. typhimurium LT2 and cloned into pET-3a vector using reported protocols.20 All mutants characterized in this study were generated by site-directed mutagenesis using the single primer method. The chimeric constructs were generated by inframe cloning of the gene segment coding for the transmembrane region of one protein into the vector containing the gene segment coding for the N-terminal helix of the other protein. E. coli BL21 (DE3) cells were transformed with the pET-3a-pagP constructs, for protein production. Proteins were expressed as inclusion bodies, and purified under denaturing conditions, as described previously.20, 23, 24

Protein refolding in lipid vesicles and detergent micelles Refolding in small unilamellar vesicles of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC,12:0 PC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, 14:0 PC) , 1,2dipalmitoyl-sn-glycero-3-phosphocholine

(DPPC,

16:0

PC),

1,2-distearoyl-sn-glycero-3-

phosphocholine (DSPC, 18:0 PC) and E. coli total lipid extract (TLE) was carried out using previously reported protocols.20 Briefly, PagP unfolded in 8.0 M guanidine hydrochloride was diluted 100-fold into a final refolding mixture containing 5 mM lipid vesicles (2.5 mg/ml in the case of total lipid extract) in 50 mM phosphate buffer pH 8.0 and 7.0 M urea at 25 °C. The final


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protein concentration was maintained at 0.08 μg/μL, which corresponded to a lipid-to-protein ratio (LPR) of ~1200:1. Refolding in n-dodecyl phosphocholine (DPC) micelles was performed as described earlier,20 by a 10-fold dilution of PagP denatured in 8.0 M urea into a refolding mixture containing 100 mM DPC in 20 mM Tris-HCl pH 9.5 at 4 °C. A 3-minute heat shock was then administered at 70 °C, followed by overnight incubation at 4 °C, to obtain refolded protein at a detergent-to-protein ratio (DPR) of ~3200:1. We examined the extent of folding in all the mutants using SDS-PAGE gel shift assay and protease protection, as described previously.20 All the PagP variants were ~100% folded.

Nuclear Magnetic Resonance (NMR) data acquisition 1

H-15N heteronuclear spin quantum coherence (HSQC) data of uniformly

15

N-labeled PagP

proteins were recorded on a Bruker Avance III 700MHz NMR instrument using a cryoprobe, as reported previously.24 PagP (~0.1 mM) refolded in 100 mM DPC containing 20 mM Tris-HCl pH 9.5 was used for HSQC measurements at 60 °C. The data were processed using NMRPipe 25 and the plots were generated using Sparky.26

Folding kinetics Folding kinetics was monitored using intrinsic tryptophan fluorescence to obtain the rates of folding for the PagP variants. Briefly, 0.6 μg/μL protein was 10-fold diluted in the folding reaction containing 10 mM DPC micelles. A λex = 295 nm was used, and emission was monitored at 340 nm for 1 h. Data was collected every 6 s to minimize photobleaching. The


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experiment dead time was ~15 s. The data was fitted to a single exponential function using the following equation: 𝑦 = 𝐴0 + 𝐴 exp(−𝑘𝑥) or a double exponential function using the following equation: 𝑦 = 𝐴0 + 𝐴1 exp(−𝑘1 𝑥) + 𝐴2 exp⁡(−𝑘2 𝑥) and the folding rates (k or k1, k2) were derived.27

Equilibrium unfolding and refolding GdnHCl–induced denaturation experiments were monitored by intrinsic tryptophan fluorescence to derive equilibrium unfolding and refolding data in DPC, as described previously.20 Unfolded or folded PagP, at a final protein concentration of 0.06 μg/μL, was treated with different GdnHCl concentrations (~0.7 M to ~8.0 M at 0.1 M increments) at 25 °C. The reaction was monitored using Trp fluorescence, recorded between 320-400 nm using a λex of 295 nm. The fluorescence emission intensity at 342 nm (λem-max) was used to calculate the unfolded fraction (𝑓𝑈 ) at each GdnHCl concentration by using the following equation:28, 29 𝑓𝑈 =

𝑦𝑂 − (𝑦𝐹 + 𝑚𝐹 [𝐷]) (𝑦𝑈 + 𝑚𝑈 [𝐷]) − (𝑦𝐹 + 𝑚𝐹 [𝐷])

Here, 𝑦𝑂 is the observed fluorescence at GdnHCl concentration [𝐷] whereas𝑦𝐹 , 𝑚𝐹 , 𝑦𝑈 and 𝑚𝑈 are intercepts and slopes of the pre- and post-transition baselines, respectively. The thermodynamic parameters, (equilibrium free energy of folding (ΔGU), m value, mid-point of chemical denaturation (𝐶𝑚 )), were derived from fits of the data to the two-state equation presented below.20, 28-31 𝑓𝑈 =

exp⁡[−(∆𝐺𝑈 + 𝑚[𝐷])/𝑅𝑇] 1 + exp⁡[−(∆𝐺𝑈 + 𝑚[𝐷])/𝑅𝑇]


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Here, R and T represent the gas constant and the experimental temperature (in K), respectively. The mid-point of chemical denaturation (𝐶𝑚 ), was calculated using the following equation: 𝐶𝑚 = ∆𝐺𝑈 /𝑚

Circular Dichroism (CD) measurements and thermal denaturation Far-UV CD scans and thermal denaturation experiments were recorded for 0.06 μg/μL protein in 10 mM DPC on a CD spectropolarimeter using a 5 mm quartz cuvette, as described previously.20, 23 Scans were recorded for the wavelength range between 207 nm and 260 nm at 10 °C, whereas thermal denaturation was monitored at 215 nm and 231 nm for a temperature range of 10-95 °C (melting) and 95-10 °C (recovery) at 1 °C increments. The CD data were converted to molar ellipticity (ME) values and a Tm-start value was calculated from linear fits to the ME231 data (ME at 231 nm).20, 32

Enzymatic activity assay The enzymatic assay was performed using a modified version of a standard lipase assay,14, 20 by utilizing p-nitrophenyl palmitate (pNPP) as the substrate to monitor the rate of product formation. In the case of enzyme assay in lipid vesicles, PagP refolded in SUVs was added to the assay mixture containing 1 mM pNPP, and 2% Triton X-100 in 50 mM phosphate buffer pH 8.0. This amounted to a final reaction mixture of 150 μL containing 1.0 μM protein and 1.2 mM lipid (0.6 mg/mL in the case of total lipid extract). For DPC-folded PagP, the same protocol was used with the following changes: the final protein and detergent concentrations were maintained at 2.0 μM and 10 mM, respectively, and the reaction was carried out in Tris-HCl pH 9.5. To derive the kinetic parameters, PagP was folded into DLPC vesicles, and assayed with increasing concentrations of the substrate, pNPP. The reaction conditions used in the case of 9 ACS Paragon Plus Environment

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lipid-refolded protein enzymatic assay were maintained here, except for the substrate concentration, which was varied from 0.05 mM to 1.5 mM. The data was fitted to the MichaelisMenten equation and the kinetic parameters, KM and Vmax, were obtained.

Database analysis Multiple sequence alignment, derived from the Pfam (pfam.sanger.ac.uk) database, was used to find out the conservation pattern for specific amino acids across 610 PagP sequences from various bacterial sources.20 Using the EVfold server,33, 34 we performed covariance analysis on 635 PagP sequences using the template sequence of PagP from E. coli (Uniprot ID: P37001), as described previously.20 For structural information related to residue contacts, the NMR structure of E. coli PagP (PDB ID: 1MM5) was used.


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RESULTS Outer membrane enzyme PagP from Salmonella and Escherichia show subtle differences in sequence The two PagP proteins from S. typhimurium (PagP-St) and E. coli (PagP-Ec) share ~83% sequence identity, with most of the differences restricted to the N-terminal helix (Fig. 1A). The role of the N-terminal helix has been investigated in E. coli PagP,14 and a general stabilizing property has been attributed to this 19-residue structure. PagP constructs that lack an N-terminal helix are considerably destabilized.14 Our previous study20 has shown that PagP-Ec shows a high Gibbs free energy of unfolding (ΔGU) as compared to PagP-St, mainly owing to an increased resistance to solvation by the denaturant (~10.5 kcal/mol for PagP-Ec; ~6.5 kcal/mol for PagPSt). In an effort to characterize the difference in stabilities of the two highly homologous PagP barrels, PagP-Ec and PagP-St, we acquired the NMR (1H-15N HSQC) spectra of both the wildtype proteins. NMR-based HSQC experiments represent an effective tool to investigate the overall structural fold of a protein as well as residue-level hydrogen bonding pattern.23,

35-37

When we compare the HSQC spectra of PagP-Ec and PagP-St refolded in DPC (n-dodecyl phosphocholine) micelles (Supplementary Material, Fig. S1), we find that both proteins show well dispersed NMR spectra, indicating well-folded structure. However, the peaks do not superpose completely. Hence, differences arising due to the N-terminal helix, which harbors considerable differences in the primary protein sequence between PagP-Ec and PagP-St, may trigger subtle structural differences between both the transmembrane scaffolds. To examine the source of the difference in thermodynamic stability between these homologs,20 we generated two chimeric constructs. For this, we interchanged the helical regions of both proteins to give PagP-EcHxSt (PagP construct containing the N-terminal helix of PagP-


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Ec and the transmembrane β-barrel domain of PagP-St) and PagP-StHxEc (PagP construct with the N-terminal helix of PagP-St and transmembrane region of PagP-Ec) (Fig. 1C). In contrast to the stability, PagP-St shows a 15-20–fold higher catalytic activity compared to the PagP-Ec barrel. This result further validated an initial observation that Salmonella membranes containing PagP show higher specific activity as compared to corresponding E. coli membranes.22 Since our analyses were carried out in membrane mimetic systems using in vitro refolded protein, the difference in enzymatic activity appears to be a function of specific residues present at or near the catalytic active site. The active site is formed at the extracellular face of the barrel, involving a catalytic triad consisting of His33, Asp76, and Ser77. The reaction has been proposed to proceed through a serine hydrolase mechanism.3 The catalytic residues, as well as those lining the substrate binding site, are identical in both PagP-Ec and PagP-St.20 Other variations in the sequence map to the transmembrane region of the barrel (Fig. 1A). Two of the most drastic mutations in the transmembrane region (L↔Q and S↔K)20 do not alter the activity of PagP by ~15-fold. A closer look at the PagP-Ec crystal structure16 suggests that Trp78 – present in the 5 Å vicinity of the active site, and is substituted by Phe in PagP-St – might play a major role in the catalysis (Fig. 1B). The aromatic Trp78 (shown in blue in Fig. 1B) appears to be in proximity to both the active site residues (green sticks) and the 12-carbon substrate analog (red spheres). We expect the interaction of W78 to be more favorable with the natural substrate for PagP, i.e., a 16-carbon palmitate chain, as established by previous studies on the hydrocarbon ruler of PagP.12, 38-40 To unravel the source of the difference in enzymatic activity by an order of magnitude between these homologs, we generated PagP-Ec W78F by mutating Trp78 to Phe, the corresponding residue in PagP-St. Similarly, we generated PagP-St F78W (Fig. 1C). Additionally, we generated


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chimeric barrels in which both modifications – N-terminal helix and aromatic residue 78 – are introduced (Fig. 1C). All constructs characterized in this study show complete folding in DPC micelles and are resistant to proteolysis (Fig. S2).

N-terminal helix modulates thermodynamic stability of outer membrane protein PagP. First, we monitored the folding rate of the wild-type PagP proteins and the chimeric barrels by following the change in tryptophan fluorescence as the protein folds in DPC micelles (Fig. 2A). We find that the folding rates of the two wild-type proteins are different (PagP-Ec: k1 ≈ 7.64 ± 1.28 s-1, k2 ≈ 0.38 ± 0.03 s-1; PagP-St: k ≈ 2.22 ± 0.09 s-1). The folding rates of the chimeric barrels PagP-StHxEc and PagP-EcHxSt (PagP-StHxEc: k1 = 6.78 ± 0.76 s-1; k2 = 0.28 ± 0.11 s-1; PagP-EcHxSt: k = 2.59 ± 0.30 s-1) are very similar to their respective wild-type barrels, PagP-Ec and PagP-St (Fig. 2A). Hence, the N-helix has little contribution to the folding rate of PagP. Our findings are in excellent agreement with previous studies.14 Similarly, neither Trp nor Phe at the 78th position influences the measured folding rate (data not shown). Equilibrium unfolding measurements of the chimeric barrels in DPC micelles reveal that exchanging the N-terminal helices leads to a switch in the stability of both proteins (Fig. 2B,C, Table 1). Specifically, PagP-EcHxSt shows a ΔGU of 11.29 ± 0.11 kcal/mol, denoting an increase of ~5 kcal/mol from wild-type PagP-St, due to the replacement of the N-terminal helix. Similarly, PagP-StHxEc shows a lowered stability of ~4 kcal/mol compared to wild-type PagPEc due to the incorporation of the helical region of PagP-St, yielding a ΔGU value of 6.52 ± 0.29 kcal/mol. We note here that this value of ΔGU for PagP-StHxEc is very similar to that of PagPSt, suggesting that the N-helix contributes considerably to PagP stability. This switch of PagP


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stability is a compound effect of changes in protein unfolding cooperativity (represented by the m value) as well as the resistance of the barrel to denaturation (changes in Cm) (Table 1). We obtained a similar inference from our far-UV CD (circular dichroism) analysis,20 wherein the secondary structure content and the tertiary interaction involving the Tyr26-Trp66 pair is qualitatively different between PagP-Ec and PagP-St (Fig. 3). The stereospecific interaction between these two residues gives rise to a CD Cotton effect with a positive maximum at ~231 nm and a negative maximum at ~225 nm.38, 39 The ME215 (molar ellipticity at 215 nm) profile of the helix swap mutant containing the helix from PagP-St fused with the barrel domain from PagP-Ec (PagP-StHxEc) shows a low value (~ –1.5 x 106 deg cm2 mol-1) resembling that of wild-type PagP-St (Fig. 3A). Similarly, the reverse mutant PagP-EcHxSt shows an increase in ME215 value compared to wild-type PagP-St, due to the incorporation of the helix from PagP-Ec. Further, thermal denaturation monitored at 215 nm (for secondary structure changes) and 231 nm (for the Tyr26-Trp66 interaction38, 39), to measure the stability of these mutants, shows us an exchange in stabilities due to the swap in N-terminal helical regions (Fig. 3B,C). The Tm-start calculated for PagP-StHxEc shows a value of 76.3 ± 2.55 °C for the unfolding reaction (Fig. 4C lower panels), which is a ~9 °C decrease compared to wild-type PagP-Ec (85.9 ± 2.27 °C). Hence, PagP-StHxEc now resembles PagP-St. A similar reversal is seen in PagP-EcHxSt, which now closely resembles PagP-Ec. Put together, our results from thermal denaturation experiments and equilibrium chemical denaturation experiments point to a crucial role for the N-terminal helix in modulating the barrel stability. The contribution of the N-terminal helix to PagP barrel stability relies heavily on favorable interactions established between the polar face of the amphipathic helix and the hydrophilic patch on the barrel (contributed by residues towards the periplasmic end of the 2nd and 3rd β-


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strands). The key residues implicated in the stability provided by the N-terminal helix, namely T16, W17 and R59,14 are conserved in both wild-type proteins (Fig. 4). Therefore, the remarkable switch in stabilities observed in the chimeric barrels appears to result from interactions of residues of the helix with the barrel, as well as those between the protein and the surrounding micelle. A detailed inspection of the sequence and structure of the amphipathic helix reveals the residues which vary across these two homologs and could collectively yield a switch in the stabilities (Fig. 4). Our thermodynamic studies from the helix swap mutants (Fig. 2 and 3) provides compelling evidence that the N-terminal helix, which was previously thought to act as a stabilizing clamp for folded PagP,14 is not merely a passive anchoring element. Through changes in the primary sequence, the N-terminal helix influences the measured stability of the transmembrane segment of PagP.

Context dependent effect on scaffold stability for aromatic residue at 78th position. We measured the change in free energy of unfolding for the Trp/Phe 78 mutants using GdnHCl–mediated equilibrium unfolding analyses. PagP-Ec W78F shows a ΔGU of 9.19 ± 0.6 kcal/mol, which corresponds to a destabilization of ~1.3 kcal/mol over the parent barrel. Similarly, the reverse mutant, F78W destabilizes the wild-type protein PagP-St by ~1.7 kcal/mol (Table 1). Since the 78th residue is located in the extracellular loop 2 of the barrel (see Fig. 1B), it does not drastically affect the hydrophobic interactions established between the protein and detergent molecules. Hence, it is surprising that a single conservative substitution brings about a destabilization of ~1.5 kcal/mol. This is a considerable change in free energy, especially when we compare with the ~0.25 kcal/mol change obtained for the more drastic W51→A mutation in PagP-Ec.

17

Additionally, the W78F mutation on PagP-Ec does not have the opposite effect as


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F78W on PagP-St. In fact, both these mutants lower the stability of the respective wild-type proteins (by ~1.5 kcal/mol; Table 1). Such an effect has been observed for several enzymes.41 It is believed that in these proteins, the active site residues can change the conformational flexibility of the local environment by virtue of being less structured. Whether the mutation of the functional residue at the 78th position destabilizes PagP by affecting the local protein dynamics is currently unclear.

Swapping of the helices does not remarkably affect the catalytic efficiency of PagP. PagP catalyzes a two-step reaction, a phospholipase reaction and a transferase reaction.12 The first step of the reaction appears to be rate limiting16,

19

and can be monitored in vitro using

different assay systems,14, 21, 22 all of which represent a test of functionality of this enzyme. One of the assay systems monitors the phospholipase reaction through p-nitrophenol release upon cleavage of the substrate analog pNPP.14 The catalytic activity of PagP-St is nearly 15-fold higher than PagP-Ec, when protein function is assayed using the lipase substrate pNPP.20 To investigate this further, we performed activity assays on the chimeric barrels refolded in the 12-C phosphocholine micelles. Contrary to the effect on stability, PagP-EcHxSt shows a specific activity of 0.35 ± 0.06 nmol/min/μM in DPC micelles, which is comparable to PagP-St.20 Similarly, PagP-StHxEc shows values of specific activity (0.015 ± 0.006 nmol/min/μM) as that of the parent barrel PagP-Ec. Hence, the N-helix does not influence PagP activity. To validate the generality of these results, we examined PagP activity in lipid vesicles of varying chain lengths. In all the lipidic systems, PagP-EcHxSt shows ~10-15–fold higher activity than PagP-StHxEc. For example, in DLPC vesicles, PagP-EcHxSt shows a specific activity of 0.95 ± 0.08 nmol/min/μM, which is similar to wild-type PagP-St.20 Similarly, the specific


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activity of PagP-StHxEc (0.04 ± 0.004 nmol/min/μM) resembles wild-type PagP-Ec. The difference in activity is faithfully reproduced also in total lipid extract obtained from E. coli, which serves as a close mimetic of the natural membrane environment of this enzyme. Here PagP-EcHxSt shows a catalytic efficiency of 0.59 ± 0.12 nmol/min/μM, which is >20-fold higher as compared to PagP-StHxEc. In line with previous results on E. coli PagP,14,

15, 18

our data

allows us to conjecture that sequence variations in the PagP helix across the homologs could evolve without considerably influencing the enzyme function.

Aromatic residue at position 78 governs differences in enzymatic activity. To test whether the aromatic residue at position 78 plays any role in the catalysis, we measured the enzymatic activity of the mutants in lipid vesicles using the standard lipase assay substrate pNPP. Surprisingly, PagP-Ec W78F shows a specific activity of 0.57 ± 0.04 nmol/min/μM in DLPC vesicles (Fig. 5A), which is nearly 10-fold higher than wild-type PagPEc. The reverse mutant, PagP-St F78W, shows a lowered specific activity of 0.07 ± 0.016 nmol/min/μM in DLPC, which is a remarkable reduction when we consider that the rest of the protein sequence (161 residues) of wild-type PagP-St is retained as-is. Other mutations near the active site [PagP-Ec T145S] or in the transmembrane region [PagP-Ec mutants L57Q,20 A71M, S91K,20 H102R] do not alter the activity considerably [data not shown]. Therefore, the conserved substitution of a single aromatic residue at position 78 has effected a dramatic change in the catalytic behavior of this enzyme. Comparison of PagP sequences from 610 organisms (Fig. 5B) indicates reasonable variation at position 78. Hence, to understand why Phe at position 78 shows higher catalytic efficiency compared to Trp, we mutated the native Trp 78 of PagP-Ec to other aromatic and aliphatic amino


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acids, and assayed the enzymatic efficiency of these mutants (Fig. 5C). In the alanine mutant (PagP-Ec W78A) the activity is nearly completely abolished, whereas substitution with the aromatic residues Tyr and His showed ~3- and ~5-fold reduction in activity, when compared with PagP-Ec W78F. Surprisingly, replacement with the hydrophobic aliphatic Leu at position 78 (PagP-Ec W78L) retained considerable PagP catalytic activity, suggesting that both aromaticity and the hydrophobicity of the 78th residue offered by Phe, may be important factors in PagP activity. The 10-fold difference in specific activity of the PagP-Ec and PagP-St chimeric mutants is reflected in activity assays performed in all the membrane mimetic systems, ranging from detergent micelles to long chain lipid vesicles, as well as E. coli total lipid extract (TLE) lacking phosphocholines (Fig. 6). For example, in TLE (bottom right panel of Fig. 6), PagP-Ec W78F shows a specific activity of 0.28 ± 0.06 nmol/min/μM, which is ~10-fold higher than PagP-Ec. Further, we also find that the lipid chain length influences the rate of pNPP hydrolysis by PagP (compare activity across all panels in Fig. 6), in line with previous findings.22,

27

The highest

activity we measure is in the 12-C and 14-C lipids DLPC and DMPC, respectively. Thinner membranes provided by 12-C and 14-C lipids are ideal mimetics for native bacterial outer membranes,42, 43 and are likely to support PagP activity. However, the influence of lipid on PagP activity is independent of the N-helix and 78th residue, as the observed change in the rate is comparable across all mutants. The observed increase in PagP-Ec W78F specific activity is approximately two-thirds of the specific activity of PagP-St in all lipids and DPC, which indicates that other molecular factors contribute to the rate of the reaction. For example, conserved substitutions (Leu↔Val or Ala↔Gly) in the loop regions or the residues lining the crenel and embrasure19 (see Fig. 1A), can account for why the activities of the wild-type proteins


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are not entirely reversed. Nevertheless, the residue that renders PagP-St more robust than PagPEc in pNPP hydrolysis appears to be the Phe at position 78. When the two modifications – the N-terminal helix and mutation of residue 78 – are exchanged and applied to the amino acid sequence of the wild-type proteins, the biophysical and biochemical properties are accordingly swapped (Fig. 1B-C, 6, Table 1). PagP-EcHxSt F78W shows high thermodynamic stability and low catalytic activity, which is uncharacteristic of the native PagP enzyme from S. typhimurium. Similarly, PagP-StHxEc W78F shows a low value of ΔGU, indicative of decreased stability, and high value of specific activity, which demonstrates increased enzymatic efficiency, both of which are properties that are not associated with E. coli PagP. The data from our chimeric constructs suggests that the properties conferred by the Nhelix and Trp/Phe 78 can act independently on PagP. In an attempt to get a better insight on the role of residue 78 in catalysis, we performed enzyme kinetics experiments on the two wild-type proteins and the site-specific W/F78 mutants. For each protein, we varied the substrate concentration from 0.05 - 1.5 mM pNPP. We experienced difficulty in dissolving the substrate pNPP beyond 1.5 mM; hence, the enzyme kinetics could not be recorded at higher pNPP concentrations. We plotted the initial rates to generate a Michaelis-Menten curve to derive the reaction kinetics. Extrapolated fits of PagP reaction kinetics gave us a qualitative estimate of the Vmax value for PagP-St (Fig. S3), which is ~12-15-fold higher than PagP-Ec and the mutant PagP-St F78W. As our enzyme kinetics are performed with the substrate analog pNPP, and within a narrow range of pNPP concentrations, the Vmax values are likely to be very different with the natural substrate. Further, the absolute values of KM and Vmax cannot be accurately obtained from our kinetics calculation. However, we anticipate that the overall trend observed in the four protein variants would be retained. Here,


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Phe appears to be more suited than Trp to enhance the catalytic rate by possibly reducing the activation barrier, rather than by promoting ease of substrate access.

Conserved 78th aromatic of PagP across bacteria suggests evolutionary importance in tailoring catalytic activity Bioinformatics analysis of the PagP sequence from 610 organisms shows that an aromatic residue is preferred at position 78 of PagP (numbering derived from PagP-Ec), across Gramnegative bacteria (Fig. 5B, 7). We performed co-evolution analysis of the PagP sequence across homologs from various bacterial sources. Surprisingly, we find that Trp 78 has a considerably high evolutionary constraint (Supplementary Material, Table S1). It is one of the 10 highestranked amino acid residues exhibiting a high evolutionary coupling (EC) score. When we take into account the structure of this protein and the spatial constraint that it imposes on the sequence, we see that the Trp 78 frequently appears in the top 100 EC pairs. Putting together the bioinformatics analysis and experimental evidence, we propose that the aromatic residue at the 78th position may be of evolutionary importance. It appears that a molecular fine-tuning of the function of PagP could have been effected through evolution through this residue, without disrupting the amino acids participating in the catalytic triad. A similar inference regarding evolutionary modulation can be derived from the coevolution analysis of the residues present in the N-terminal helix, although further experimental validation is required to assert this claim. We find that the sequence variation is highest in the Nterminal helical region (Fig. 7). Additionally, several high-scoring residues represented in the EC Hotspot table (Table S1) belong to the helix. The evolutionary network diagram (Fig. S4) shows that the N-terminal helical residues variant across PagP-Ec and PagP-St are indeed under tight


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evolutionary control. If the regulation of PagP stability in vivo is reliant on the N-terminal helical sequence, as seen from in vitro experiments, it could have been brought about by co-evolutionary mechanisms. Such mechanisms could either be due to directed evolution of these proteins to accommodate the PagP barrel better in their respective outer membrane environments, or compensatory mutations in the mature protein sequence arising from the high mutational frequency of the signal peptide.44


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DISCUSSION The lipid A palmitoyltransferase enzyme PagP resides in the asymmetric lipid bilayer of the Gram-negative bacterial outer membrane and confers bacterial resistance by modifying the lipid composition of the endotoxin moiety.9,

11, 45, 46

Although this protein has been previously

characterized in detail, the exact molecular factors responsible for its structural stability and catalytic ability still remain unresolved. In this study, through mutational analysis of nearly identical homologs from E. coli and S. typhimurium, we address this question and provide compelling evidence to prove that the two molecular features – thermodynamic stability and catalytic efficiency – are modulated by seemingly distinct regions of PagP (Fig. 7). On the one hand, the N-terminal helix dictates the stability of the barrel by providing more favorable (PagPEc) or less favorable (PagP-St) interactions at the docking interface. However, the N-helix neither alters the refolding rate of the barrel,14 nor the catalytic activity.15, 18 On the other hand, the single aromatic residue (Trp for PagP-Ec and Phe for PagP-St), placed diametrically opposite to the N-terminal helix in the L2 loop, modulates the catalytic efficiency possibly by influencing the rate of product formation. The overall modulators of stability and function are demarcated in PagP, and involve a minimal set of mutations at the N-terminal helix and the 78th residue. Hence, our PagP-EcHxSt (PagP-Ec N-terminal helix with PagP-St barrel) is the most stable barrel with the highest activity. An important inference from the chimeric barrels is that the effects of these two modifications on the thermodynamic stability are non-additive. Therefore, it appears that the N-helix and the 78th residue are coupled and cannot be considered as entirely distinct. Indeed, our analysis reveals the existence of a networked interaction effected by subtle changes in the residues joining the structural (helix) and functional (active site) elements of PagP (Fig. 7 and S4). Further, we find


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that the lipid chain length influences the activity of both PagP-Ec and PagP-St (Fig. 6), as speculated earlier.22,

27

How well our in vitro findings resemble in vivo PagP behavior still

remains to be tested, as our measurements of protein stability in DPC micelles can be vastly different from the asymmetric lipopolysaccharide environment of the bacterial outer membrane. Yet, there is reasonable belief that in vitro results can be extended to native conditions.47,

48

Further, our enzymatic assay provides a qualitative insight only into the first step of the two-step reaction catalyzed by PagP. While the first step is considered rate-limiting,16, 19 whether the aryl ring does contribute to the transferase activity of PagP would constitute an interesting study. Changes to the active site residues (His 33, Asp 76, Ser 77) inactivates PagP.3 Hence, it is tempting to speculate that substitutions of residues proximal to the active site, such as Trp/Phe 78, can alter the phospholipase activity of PagP. The importance of the 78th position in PagP function is also supported by the effect that mutations at this site have on the barrel thermodynamic stability. Such a tradeoff between fold and function has been observed for many enzymes.49-52 It is also known that functionally relevant sites tend to reduce the foldability of the protein, by introducing local structural disorder.41 A structurally flexible barrel is important for substrate and product diffusion in PagP19,

21

and multiple structural states are known for this

protein from NMR21 and folding17 studies. The lowered stability of the Trp/Phe 78 mutants might be due to a change in the local protein dynamicity. However, the modeled PagP-Ec structure with Phe78 forms similar non-covalent contacts in its 5 Å vicinity as the wild-type protein with Trp78 (not shown). Therefore, the difference could arise from altered interaction strengths of Trp and Phe with the surrounding lipid environment. In the bacterial outer membrane, the interaction of the protein with lipids of the outer leaflet may also dictate the


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Page 24 of 51

structural and functional dynamics of this enzyme.7, 10, 22 We anticipate that mutational studies in vivo will shed more light on the importance of Trp/Phe 78 in both bacteria. Under physiological conditions, PagP expression levels are low and is upregulated during stress.3,

7, 10, 47

Further, PagP protein level in E. coli outer membranes is less than that in

Salmonella.7, 22 Differences in PagP stability and possible differences in catalytic efficiency can together mirror the adaptation of both organisms to their respective environments.7 Indeed, a biochemical difference in both mature proteins is considered one of the likely factors for the robustness of Salmonella PagP over its Escherichia homolog.7,

22

Co-evolutionary pressure,

customized functional requirements of PagP in the outer membrane,7 barrel stability,20 contributions of chaperones and accessory factors,17,

53-55

and changes in the membrane

composition6 can together decide the residence time of PagP in the membrane during stress and protein turnover under physiological or stress conditions. Our results provide insight on the distinct factors that can govern the evolution of membrane proteins, and the role of select residues in PagP function in the bacterial outer membrane.


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Acknowledgements B.R.I is a senior research fellow of the University Grants Commission, Govt. of India. R.M. is a Wellcome Trust/DBT India Alliance Intermediate Fellow.

Funding This work was supported by the Department of Biotechnology Grants BT/HRD/35/02/25/2009, BT/01/IYBA/2009 from the Government of India to R.M.

Conflict of Interest The authors declare that they have no conflict of interest with the contents of this article.

Author Contributions R.M. and B.R.I. conceived the study. B.R.I. performed the experiments. Both authors analyzed the data and wrote the paper.

Supplementary Material Supplementary Material is available with the online version of this article.


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Wang, X., Minasov, G., and Shoichet, B. K. (2002) Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs, J. Mol. Biol. 320, 85-95. Tokuriki, N., Stricher, F., Serrano, L., and Tawfik, D. S. (2008) How protein stability and new functions trade off, PLoS Comput. Biol. 4, e1000002. Soskine, M., and Tawfik, D. S. (2010) Mutational effects and the evolution of new protein functions, Nat. Rev. Genet. 11, 572-582. Studer, R. A., Christin, P. A., Williams, M. A., and Orengo, C. A. (2014) Stabilityactivity tradeoffs constrain the adaptive evolution of RubisCO, Proc. Natl. Acad. Sci. U. S. A. 111, 2223-2228. Hagan, C. L., Silhavy, T. J., and Kahne, D. (2011) beta-Barrel membrane protein assembly by the Bam complex, Annu. Rev. Biochem. 80, 189-210. McMorran, L. M., Bartlett, A. I., Huysmans, G. H., Radford, S. E., and Brockwell, D. J. (2013) Dissecting the effects of periplasmic chaperones on the in vitro folding of the outer membrane protein PagP, J. Mol. Biol. 425, 3178-3191. McMorran, L. M., Brockwell, D. J., and Radford, S. E. (2014) Mechanistic studies of the biogenesis and folding of outer membrane proteins in vitro and in vivo: what have we learned to date?, Arch. Biochem. Biophys. 564, 265-280. http://www.ebi.ac.uk/Tools/msa/clustalw2/. Crooks, G. E., Hon, G., Chandonia, J. M., and Brenner, S. E. (2004) WebLogo: a sequence logo generator, Genome Res. 14, 1188-1190.


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TABLE Table 1. Summary of thermodynamic parameters for PagP variants. PagP-Ec a ΔGU

PagP-St a m

ΔGU

Cm

m

Cm

Protein kcal mol- kcal mol-

kcal mol- kcal molM

1

10.51 Ec < > St

1

M

M-1

1

± -3.05

± 3.45

±

6.46

1

M-1

± -2.28

± 2.83

±

b

0.47 6.52

0.14

0.15

± -2.26

± 2.89

0.28 ±

11.29

0.09 ± -3.82

0.11 ± 2.96

±

StHxEc < > EcHxSt 0.29 9.19

0.15

0.07

± -2.91

± 3.18

0.11 ±

4.72

0.19 ± -1.59

0.13 ± 2.96

±

W78F < > F78W 0.61 StHxEc

W78F

EcHxSt F78W a

7.28 0.29

0.35

0.17

± -2.49

± 2.92

0.12

s.d. from three independent experiments.

0.21 ±

0.03 b

10.79 0.79

0.06 ± -3.39 0.33

0.05 ± 3.19 0.07

Data for wild-type PagP-Ec and PagP-St are

reproduced here with permission from Iyer and Mahalakshmi, Biochemistry, 2015, 54, 57125722. Copyright (2015) American Chemical Society.


±

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Biochemistry

FIGURE LEGENDS Figure 1. Pictorial representation of the proteins characterized in this study. (A) Ribbon diagram of PagP-Ec (orange) generated from the crystal structure (PDB ID: 3GP6). Residues associated with catalysis are shown as green sticks, the N-terminal helix is rendered in cyan and W78, discussed in our study, is shown as blue spheres. Residue variations that represent conservative substitutions in the loop regions or the barrel exterior are shown as yellow sticks. (B) Cartoon representation of PagP-Ec showing a sodium dodecyl sulfate molecule (red spheres) trapped in the substrate binding cavity formed by the protein (orange ribbon). The active site residues are rendered as green sticks whereas the aromatic Trp78 is rendered as blue spheres. The distance between the center of the indole ring and the headgroup of SDS is indicated. (C) Schematic figure showing PagP displayed as an integral membrane barrel and the N-terminal helix as embedded in the membrane. PagP-Ec (red), PagP-St (blue) and all the mutants described in this study, namely the helix swap mutants (PagP-StHxEc, PagP-EcHxSt), mutation at the 78th residue (shown as crescent shape on the barrel) and helix swap mutants generated on a W/F-78 background (extreme right), are colored according to parent protein sequence. All the constructs are displayed in a membrane bilayer solely for illustration.

Figure 2. Equilibrium unfolding profiles of helix swap mutants. (A) Folding kinetics monitored using tryptophan fluorescence in DPC micelles. Shown here are representative traces for (left) PagP-Ec, PagP-StHxEc and (right) PagP-St, PagP-EcHxSt. Fits of the data to an exponential function to derive folding rates are shown as solid and dotted lines. (B) Representative fluorescence emission profiles of PagP-StHxEc (left) and PagP-EcHxSt (right) derived from GdnHCl-induced chemical denaturation from low (purple) to high (red) denaturant


Biochemistry

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concentration. A.U.: arbitrary units. (C) Unfolded fractions (fU) calculated for PagP-StHxEc (left, brown circles) and PagP-EcHxSt (right, blue circles) fitted to a two-state equation (fits shown as solid lines). Standard deviation was calculated from three independent experiments. The equilibrium unfolding fits for the wild-type proteins20 are represented as dashed lines (Ecred, St-blue) in both panels, for comparison. Data for wild-type PagP-Ec and PagP-St are reproduced here with permission from Iyer and Mahalakshmi, Biochemistry, 2015, 54, 57125722. Copyright (2015) American Chemical Society.

Figure 3. Far-UV CD analyses of the helix swap mutants. (A) Far-UV CD scans of PagPStHxEc (brown triangles) and PagP-EcHxSt (oxford blue triangles) refolded in 10 mM DPC are superposed with wild-type PagP-Ec (red empty circles) and PagP-St (blue empty circles)

20

data

showing both signature peaks of PagP at ~218 nm and ~231 nm. (B) Tm-start values, derived from thermal denaturation and recovery experiments monitored by CD at 231 nm, are tabulated. Data for wild-type PagP-Ec and PagP-St are reproduced here with permission from Iyer and Mahalakshmi, Biochemistry, 2015, 54, 5712-5722. Copyright (2015) American Chemical Society. (C) Representative thermal denaturation (filled symbols) and recovery (empty symbols) profiles are shown for PagP-StHxEc (left panels) and PagP-EcHxSt (right panels). The ME231 data (bottom panels) were used for calculation of Tm-start shown in (B).

Figure 4. Cartoon representation of N-terminal amphipathic helix highlighting sequence differences that can demarcate stability. (A, left) Structural rendering of the N-terminal helix (obtained from PagP-Ec NMR structure; PDB ID: 1MM4), displaying the amphipathic nature of the motif consisting of distinct polar (blue) and apolar (orange) surfaces. The two residues


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Biochemistry

essential for providing stability, namely W17 (which completes the aromatic girdle) and T16 (which forms favorable helix-barrel interaction with R59, an arginine residue present in strand 2 of PagP)

14

are represented as sticks. (A, middle and right) Residues present in the N-terminal

helix that are variant across PagP-Ec and PagP-St are shown as spheres. Many of these are present on the polar face of the helix (in blue). (B) ClustalW

56

alignment of the 19-residue N-

terminal helix from E. coli K12 (Ec) and S. typhimurium LT2 (St) showing sequence variation along the length of the helix. Residues are colored according to side chain properties as blue (polar) and orange (apolar).

Figure 5. Catalytic efficiency of single aromatic substituents of PagP compared with parent proteins. (A) Enzymatic assay of PagP refolded in DLPC vesicles towards the substrate analogue pNPP. (Left) Increase in A405 upon enzymatic release of p-nitrophenol is plotted against time for PagP-Ec (red circles) and its mutant W78F (dark red triangles). (Right) A decrease in A405 is seen for PagP-St F78W (dark blue triangles) compared to its parent protein PagP-St (blue circles). Error bars are derived from two independent experiments. (B) Multiple sequence alignment of PagP obtained from the Pfam database (pfam.sanger.ac.uk) is presented in WebLogo format57 for residues 72–84. (C) Enzyme activity assay of PagP-Ec mutated with different representative aromatic and aliphatic residues at position 78. Specific activity is calculated based on A405 increase upon hydrolysis of the substrate analogue pNPP by 2.0 μM protein refolded in n-dodecyl phosphocholine (DPC) micelles (see Experimental Procedures for details). Abscissa are labeled according to the single letter code of the residue present at position 78 in the mutant, starting with PagP-Ec wild-type containing W. Assays for W78Y, W78H, W78L and W78A were performed using inclusion bodies prepared using reported methods.24


Biochemistry

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Data for PagP-Ec (in (A) and (C)) and PagP-St (in (A)) is reproduced here with permission from Iyer and Mahalakshmi, Biochemistry, 2015, 54, 5712-5722. Copyright (2015) American Chemical Society. Data is represented as a mean of at least two independent experiments.

Figure 6. Comparing enzymatic activity in different membrane mimetic environments for all the variants described in this study. Specific enzyme activity is compared across different mutants refolded in membrane mimetics ranging from DPC micelles to vesicles comprising of 12-C (DLPC), 14-C (DMPC), 16-C (DPPC), 18-C (DSPC) and total lipid extract (TLE) obtained from E. coli membranes. Specific activity is calculated based on A405 increase upon hydrolysis of the substrate analogue pNPP by 2.0 μM protein (see Experimental Procedures for details). Abscissa labels: Ec: PagP-Ec, bright red; St: PagP-St, bright blue; SHE: PagP-StHxEc, brown; EHS: PagP-EcHxSt, oxford blue; W78F: PagP-Ec W78F, maroon; F78W: PagP-St F78W, navy blue; SHEW: PagP-StHxEc W78F, orange; EHSF: PagP-EcHxSt F78W, pale blue. Data for wild type PagP-Ec and PagP-St are reproduced here for comparison, with permission from Iyer and Mahalakshmi, Biochemistry, 2015, 54, 5712-5722. Copyright (2015) American Chemical Society.

Figure 7. Schematic summarizing the independent modulators of PagP activity and stability. Multiple sequence alignment of PagP from five different bacterial sources is depicted for the N-terminal helix (left) and the region containing W78 (right). Residues that are identical across the five bacteria are highlighted in yellow whereas residues which are variant but represent conservative substitutions are highlighted in green. Shown in the middle is the ribbon


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Biochemistry

diagram of PagP-Ec (orange) generated from the crystal structure (PDB ID: 3GP6) with the Nterminal helix rendered in cyan, and W78, identified in our study, shown as blue spheres. Residues that coevolve with both the N-helix and W78 are shown as grey sticks and are connected through dotted lines.


Biochemistry

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FIGURES Figure 1.


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Biochemistry

Figure 2.


Biochemistry

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Figure 3.


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Biochemistry

Figure 4.


Biochemistry

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Figure 5.


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Biochemistry

Figure 6.


Biochemistry

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Figure 7.


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Biochemistry

Table of Contents Graphic


Biochemistry

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Figure 1. Pictorial representation of the proteins characterized in this study. Fig. 1 101x61mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Biochemistry

Figure 2. Equilibrium unfolding profiles of helix swap mutants. Fig. 2 129x197mm (300 x 300 DPI)


Biochemistry

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Figure 3. Far-UV CD analyses of the helix swap mutants. Fig. 3 83x81mm (300 x 300 DPI)


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Biochemistry

Figure 4. Cartoon representation of N-terminal amphipathic helix highlighting sequence differences that can demarcate stability. Fig. 4 72x62mm (300 x 300 DPI)


Biochemistry

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Figure 5. Catalytic efficiency of single aromatic substituents of PagP compared with parent proteins. Fig. 5 143x241mm (300 x 300 DPI)


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Biochemistry

Figure 6. Comparing enzymatic activity in different membrane mimetic environments for all the variants described in this study. Fig. 6 89x50mm (300 x 300 DPI)


Biochemistry

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Figure 7. Schematic summarizing the independent modulators of PagP activity and stability. Fig. 7 65x23mm (300 x 300 DPI)


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Biochemistry

Table of Contents Graphic Table of Contents Graphic 35x14mm (300 x 300 DPI)


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