Disorder is a critical component of lipoprotein sorting in Gram-negative bacteria 1 Disorder is a critical component of lipoprotein sorting in Gram-negative bacteria 1 2 Jessica El Rayes1,2$, Joanna Szewczyk1,2$, Michael Deghelt1,2, André Matagne3, Bogdan I. 3 Iorga4, Seung-Hyun Cho1,2, and Jean-François Collet1,2* 4 5 6 1WELBIO, Avenue Hippocrate 75, 1200 Brussels, Belgium. 7 2de Duve Institute, Université catholique de Louvain, Avenue Hippocrate 75, 1200 Brussels, 8 Belgium. 9 3Centre d’ingéniérie des Protéines, Institut de Chimie B6, Université de Liège, Allée de la 10 Chimie 3, 4000 Liège, Sart Tilman, Belgium. 11 4Université Paris-Saclay, CNRS UPR 2301, Institut de Chimie des Substances Naturelles, 12 91198 Gif-sur-Yvette, France. 13 14 $Both authors contributed equally to the work 15 16 *Correspondence: jfcollet@uclouvain.be 17 2 Abstract (150 max) 18 19 Gram-negative bacteria express structurally diverse lipoproteins in their envelope. Here 20 we found that approximately half of lipoproteins destined to the Escherichia coli outer 21 membrane display an intrinsically disordered linker at their N-terminus. Intrinsically 22 disordered regions are common in proteins, but establishing their importance in vivo has 23 remained challenging. Here, as we sought to unravel how lipoproteins mature, we 24 discovered that unstructured linkers are required for optimal trafficking by the Lol 25 lipoprotein sorting system: linker deletion re-routes three unrelated lipoproteins to the 26 inner membrane. Focusing on the stress sensor RcsF, we found that replacing the linker 27 with an artificial peptide restored normal outer membrane targeting only when the 28 peptide was of similar length and disordered. Overall, this study reveals the role played 29 by intrinsic disorder in lipoprotein sorting, providing mechanistic insight into the 30 biogenesis of these proteins and suggesting that evolution can select for intrinsic disorder 31 that supports protein function. 32 3 Introduction 33 The cell envelope is the morphological hallmark of Escherichia coli and other Gram-negative 34 bacteria. It is composed of the inner membrane, a classical phospholipid bilayer, as well as the 35 outer membrane, an asymmetric bilayer with phospholipids in the inner leaflet and 36 lipopolysaccharides in the outer leaflet1. This lipid asymmetry enables the outer membrane to 37 function as a barrier that effectively prevents the diffusion of toxic compounds in the 38 environment into the cell. The inner and outer membranes are separated by the periplasm, a 39 viscous compartment that contains a thin layer of peptidoglycan also known as the cell wall1. 40 The cell envelope is essential for growth and survival, as illustrated by the fact that several 41 antibiotics such as the b-lactams target mechanisms of envelope assembly. Mechanisms 42 involved in envelope biogenesis and maintenance are therefore attractive targets for novel 43 antibacterial strategies. 44 45 Approximately one-third of E. coli proteins are targeted to the envelope, either as soluble 46 proteins present in the periplasm or as proteins inserted in one of the two membranes2. While 47 inner membrane proteins cross the lipid bilayer via one or more hydrophobic α-helices, proteins 48 inserted in the outer membrane generally adopt a β-barrel conformation3. Another important 49 group of envelope proteins is the lipoproteins, which are globular proteins anchored to one of 50 the two membranes by a lipid moiety. Lipoproteins carry out a variety of important functions 51 in the cell envelope: they participate in the biogenesis of the outer membrane by inserting 52 lipopolysaccharide molecules4,5 and b-barrel proteins6, they function as stress sensors triggering 53 signal transduction cascades when envelope integrity is altered7, and they control processes that 54 are important for virulence8. The diverse roles played by lipoproteins in the cell envelope has 55 drawn a lot of attention lately, revealing how crucial these proteins are in a wide range of vital 56 processes and identifying them as attractive targets for antibiotic development. Yet, a detailed 57 4 understanding of the mechanisms involved in lipoprotein maturation and trafficking is still 58 missing. 59 60 Lipoproteins are synthesized in the cytoplasm as precursors with an N-terminal signal peptide9. 61 The last four C-terminal residues of this signal peptide, known as the lipobox, function as a 62 molecular determinant of lipid modification unique to bacteria; only the cysteine at the last 63 position of the lipobox is strictly conserved10. After secretion of the lipoprotein into the 64 periplasm, the thiol side-chain of the cysteine is first modified with a diacylglyceryl moiety by 65 prolipoprotein diacylglyceryl transferase (Lgt)9 (Extended Data Fig. 1a, step 1). Then, signal 66 peptidase II (LspA) catalyzes cleavage of the signal peptide N-terminally of the lipidated 67 cysteine before apolipoprotein N-acyltransferase (Lnt) adds a third acyl group to the N-terminal 68 amino group of the cysteine (Extended Data Fig. 1a, steps 2-3). Most mature lipoproteins are 69 then transported to the outer membrane by the Lol system. Lol consists of LolCDE, an ABC 70 transporter that extracts lipoproteins from the inner membrane and transfers them to the soluble 71 periplasmic chaperone LolA (Extended Data Fig. 1a, steps 4-5)11. LolA escorts lipoproteins 72 across the periplasm, binding their hydrophobic lipid tail, and delivers them to the outer 73 membrane lipoprotein LolB (Extended Data Fig. 1a, step 6). LolB finally anchors lipoproteins 74 to the inner leaflet of the outer membrane using a mechanism that remains poorly characterized 75 (Extended Data Fig. 1a, step 7). 76 77 In most Gram-negative bacteria, a few lipoproteins remain in the inner membrane12,13. The 78 current view is that inner membrane retention depends on the identity of the two residues 79 located immediately downstream of the N-terminal cysteine on which the lipid moiety is 80 attached14; this sequence, two amino acids in length, is known as the Lol sorting signal. When 81 lipoproteins have an aspartate at position +2 and an aspartate, glutamate, or glutamine at 82 5 position +3, they remain in the inner membrane15,16, possibly because strong electrostatic 83 interactions between the +2 aspartate and membrane phospholipids prevent their interaction 84 with LolCDE17. However, this model is largely based on data obtained in E. coli and variations 85 have been described in other bacteria. For instance, in the pathogen Pseudomonas aeruginosa, 86 an aspartate is rarely found at position +2 and inner membrane retention appears to be 87 determined by residues +3 and +418,19. Surprisingly, lipoproteins are well sorted in P. 88 aeruginosa cells expressing the E. coli LolCDE complex20, despite their different Lol sorting 89 signal. This result cannot be explained by the current model of lipoprotein sorting, underscoring 90 that our comprehension of the precise mechanism that governs the triage of lipoproteins remains 91 incomplete. 92 93 Excitingly, more unresolved questions regarding lipoprotein biogenesis have recently been 94 raised. First, it was reported that a LolA-LolB-independent trafficking route to the outer 95 membrane exists in E. coli21, but the factors involved have remained unknown. Second, 96 although lipoproteins have traditionally been considered to be exposed to the periplasm in E. 97 coli and many other bacterial models9, a series of investigations have started to challenge this 98 view by identifying lipoproteins on the surface of E. coli, Vibrio cholerae, and Salmonella 99 Typhimurium22-26. Overall, the field is beginning to explore a lipoprotein topological landscape 100 that is more complex than previously assumed and raising intriguing questions about the signals 101 that control surface targeting and exposure. 102 103 Here, stimulated by the hypothesis that crucial details of the mechanisms underlying lipoprotein 104 maturation remained to be elucidated, we sought to identify novel molecular determinants 105 controlling lipoprotein biogenesis. First, we systematically analyzed the sequence of the 66 106 lipoproteins with validated localization27 encoded by the E. coli K12 genome27 and found that 107 6 half of the outer membrane lipoproteins display a long and intrinsically disordered linker at 108 their N-terminus. Intrigued by these unstructured segments, we then probed their importance 109 for the biogenesis of RcsF, NlpD, and Pal, three structurally and functionally unrelated outer 110 membrane lipoproteins. Unexpectedly, we found that deleting the linker—while keeping the 111 Lol sorting signal intact—altered the targeting of all three lipoproteins to the outer membrane, 112 with physiological consequences. Focusing on RcsF, we determined that both the length and 113 disordered character of the linker were important. Remarkably, lowering the load of the Lol 114 system by deleting lpp, which encodes the most abundant lipoprotein (~1 million copies per 115 cell28), restored normal outer membrane targeting of linker-less RcsF, indicating that the N-116 terminal linker is required for optimal lipoprotein processing by Lol. Taken together, these 117 observations reveal the unsuspected role played by protein intrinsic disorder in lipoprotein 118 biogenesis. 119 7 Results 120 121 Half of E. coli lipoproteins present long disordered segments at their N-termini 122 In an attempt to discover novel molecular determinants controlling the biogenesis of 123 lipoproteins, we decided to systematically analyze the sequence of the lipoproteins encoded by 124 the E. coli genome (strain MG1655) in search of unidentified structural features. E. coli encodes 125 ~80 validated lipoproteins29, of which 58 have been experimentally shown to localize in the 126 outer membrane27. Comparative modeling of existing X-ray, cryogenic electron microscopy 127 (cryo-EM), and nuclear magnetic resonance (NMR) structures revealed that approximately half 128 of these outer membrane lipoproteins display a long segment (>22 residues) that is predicted to 129 be disordered at the N-terminus (Fig. 1, Extended Data Fig. 2, Extended Data Table 1). In 130 contrast, only one of the 8 lipoproteins that remain in the inner membrane (DcrB; Extended 131 Data Fig. 2, Extended Data Table 1) had a long, disordered linker, suggesting that disordered 132 peptides may be important for lipoprotein sorting. 133 134 Deleting the N-terminal linker of RcsF, NlpD, and Pal perturbs their targeting to the outer 135 membrane 136 Intrigued by the presence of these N-terminal disordered segments in so many outer membrane 137 lipoproteins, we decided to investigate their functional importance. We selected three 138 structurally unrelated lipoproteins whose function could easily be assessed: the stress sensor 139 RcsF (which triggers the Rcs signaling cascade when damage occurs in the envelope30), NlpD 140 (which activates the periplasmic N-acetylmuramyl-L-alanine amidase AmiC, which is involved 141 in peptidoglycan cleavage during cell division31,32), and the peptidoglycan-binding lipoprotein 142 Pal (which is important for outer membrane constriction during cell division33). 143 144 8 We began by preparing truncated versions of RcsF, NlpD, and Pal devoid of their N-terminal 145 unstructured linkers (Extended Data Fig. 1b, Extended Data Fig. 2; RcsF∆19-47, Pal∆26-56, and 146 NlpD∆29-64). Note that the lipidated cysteine residue (+1) and the Lol sorting signal (the amino 147 acids at positions +2 and +3) were not altered in RcsF∆19-47, Pal∆26-56, and NlpD∆29-64, nor in any 148 of the constructs discussed below (Extended Data Table 2). For Pal, although the unstructured 149 linker spans residues 25-68 (Fig. 1), we used Pal∆26-56 because Pal∆25-68 was either degraded or 150 not detected by the antibody (data not shown). We first tested whether the truncated lipoproteins 151 were still correctly extracted from the inner membrane and transported to the outer membrane. 152 The membrane fraction was prepared from cells expressing the three variants independently, 153 and the outer and inner membranes were separated using sucrose density gradients (Methods). 154 Whereas wild-type RcsF, NlpD, and Pal were mostly detected (>90%) in the outer membrane 155 fraction, as expected, ~50% of RcsF∆19-47 and ~60% of NlpD∆29-64 were retained in the inner 156 membrane (Fig. 2a, 2b). The sorting of Pal was also affected, although to a lesser extent: 15% 157 of Pal∆26-56 was retained in the inner membrane (Fig. 2c). Notably, the expression levels of the 158 three linker-less variants were similar (NlpD∆29-64) or lower (RcsF∆19-47; Pal∆26-56) than those of 159 the wild-type proteins (Extended Data Fig. 3), indicating that accumulation in the inner 160 membrane did not result from increased protein abundance. 161 162 We then tested the impact of linker deletion on the function of these three proteins. In cells 163 expressing RcsF∆19-47, the Rcs system was constitutively turned on (Fig. 2d); when RcsF 164 accumulates in the inner membrane, it becomes available for interaction with IgaA, its 165 downstream Rcs partner in the inner membrane30,34. Likewise, expression of NlpD∆29-64 did not 166 rescue the chaining phenotype (Fig. 2e)35 exhibited by cells lacking both nlpD and envC, an 167 activator of the amidases AmiA and AmiB32. Finally, Pal∆26-56 partially rescued the sensitivity 168 of the pal mutant to SDS-EDTA that results from increased membrane permeability36 (Fig. 2f). 169 9 However, this observation needs to be considered with caution given that Pal∆26-56 seemed to 170 be expressed at lower levels than wild-type Pal (Extended Data Fig. 3). Thus, preventing 171 normal targeting of RcsF, NlpD and Pal to the outer membrane had functional consequences. 172 173 RcsF variants with unstructured artificial linkers of similar lengths are normally targeted 174 to the outer membrane 175 The results above were surprising because they revealed that the normal targeting of RcsF, 176 NlpD, and Pal to the outer membrane does not only require an appropriate Lol sorting signal, 177 as proposed by the current model for lipoprotein sorting9, but also the presence of an N-terminal 178 linker. We selected RcsF, whose accumulation in the inner membrane can be easily tracked by 179 monitoring Rcs activity30,37, to investigate the structural features of the linker controlling 180 lipoprotein maturation; keeping as little as 10% of the total pool of RcsF molecules in the inner 181 membrane is sufficient to fully activate Rcs30. 182 183 We first tested whether changing the sequence of the N-terminal segment while preserving its 184 disordered character still yielded normal targeting of the protein to the outer membrane. To that 185 end, we prepared an RcsF variant in which the N-terminal linker was replaced by an artificial, 186 unstructured sequence (Extended Data Table 2, Extended Data Fig. 2, Extended Data Fig. 187 4) of similar length and consisting mostly of GS repeats (RcsFGS). Substituting the wild-type 188 linker with this artificial sequence was remarkably well tolerated by RcsF: RcsFGS was targeted 189 normally to the outer membrane (Fig. 3a) and did not constitutively activate the stress system 190 (Fig. 3b). Thus, although RcsFGS has an N-terminus with a completely different primary 191 structure, it behaved like the wild-type protein. 192 193 10 We then investigated whether the N-terminal linker required a minimal length for proper 194 targeting and function. We therefore constructed two RcsF variants with shorter, unstructured, 195 artificial linkers (RcsFGS2 and RcsFGS3, with linkers of 18 and 10 residues, respectively; 196 Extended Data Table 2, Extended Data Fig. 2, Extended Data Fig. 4). Importantly, RcsFGS2 197 and, to a greater extent, RcsFGS3 did not properly localize to the outer membrane: the shorter 198 the linker, the more RcsF remained in the inner membrane (Fig. 3a). Consistent with the amount 199 of RcsFGS2 and RcsFGS3 retained in the inner membrane, Rcs activation levels were inversely 200 related to linker length (Fig. 3b). 201 202 The disordered character of the linker is required for normal targeting 203 Taken together, the results above demonstrated that the RcsF linker can be replaced with an 204 artificial sequence lacking secondary structure, provided that it is of appropriate length. Next, 205 we sought to directly probe the importance of having a disordered linker by replacing the RcsF 206 linker with an alpha-helical segment 35 amino acids long from the periplasmic chaperone FkpA 207 (RcsFFkpA; Extended Data Table 2, Extended Data Fig. 2, Extended Data Fig. 4). 208 Introducing order at the N-terminus of RcsF dramatically impacted the protein distribution 209 between the two membranes: RcsFFkpA was substantially retained in the inner membrane (Fig. 210 3c) and constitutively activated Rcs (Fig. 3d). As alpha-helical segments are considerably 211 shorter than unstructured sequences containing a similar number of amino acids, we also 212 prepared an RcsF variant (RcsFcol) with a longer alpha helix from the helical segment of colicin 213 Ia, which is 73 amino acids in length and also predicted to remain folded in the RcsFcol construct 214 (Extended Data Table 2, Extended Data Fig. 2, Extended Data Fig. 4). However, doubling 215 the size of the helix had no impact, with RcsFcol behaving similarly to RcsFFkpA (Fig. 3c, 3d). 216 Together, these data demonstrate that having an N-terminal disordered linker downstream of 217 the Lol sorting signal is required to correctly target RcsF to the outer membrane. The length of 218 11 the linker is important, but the sequence is not, on the condition that the linker does not fold 219 into a defined secondary structure. 220 221 The disordered linker is required for optimal processing by Lol 222 Our finding that N-terminal disordered linkers function as molecular determinants of the 223 targeting of lipoproteins to the outer membrane raised the question of whether these linkers 224 work in a Lol-dependent or Lol-independent manner. To address this mechanistic question, we 225 tested the impact of deleting lpp on the targeting of RcsF∆19-47. The lipoprotein Lpp, also known 226 as the Braun lipoprotein, covalently tethers the outer membrane to the peptidoglycan and 227 controls the size of the periplasm38,39. Being expressed at ~1 million copies per cell28, Lpp is 228 numerically the most abundant protein in E. coli. Thus, by deleting lpp, we considerably 229 decreased the load on the Lol system by removing its most abundant substrate. Remarkably, 230 lpp deletion fully rescued the targeting of RcsF∆19-47 to the outer membrane (Fig. 4a), indicating 231 that the linker functions in a Lol-dependent manner and suggesting that accumulation of 232 RcsF∆19-47 in the inner membrane results from a decreased ability of the Lol system to process 233 the linker-less RcsF variant. Importantly, similar results were obtained with NlpD∆29-64, which 234 was also correctly targeted to the outer membrane in cells lacking Lpp (Fig. 4a). Pal∆26-56 could 235 not be tested because membrane fractionation failed with lpp pal double mutant cells whether 236 or not they expressed Pal∆26-56 (data not shown). 237 238 To obtain further insights into the mechanism at play here, we next monitored whether linker 239 deletion impacted the transfer of RcsF from LolA to LolB in vitro. LolA with a C-terminal His-240 tag was expressed in the periplasm of cells expressing wild-type RcsF or RcsF∆19-47 and purified 241 to near homogeneity via affinity chromatography (Methods; Extended Data Fig. 5). Both RcsF 242 and RcsF∆19-47 were detected in immunoblots of the fractions containing purified LolA 243 12 (Extended Data Fig. 5), indicating that both proteins form a soluble complex with LolA and 244 confirming that they use this chaperone for transport across the periplasm. LolB was expressed 245 as a soluble protein in the cytoplasm and purified by taking advantage of a C-terminal Strep-246 tag; LolB was then incubated with LolA-RcsF or LolA-RcsF∆19-47 and pulled-down using 247 Streptactin beads (Methods). As both RcsF and RcsF∆19-47 were detected in the LolB-containing 248 pulled-down fractions (Fig. 4b), we conclude that both proteins were transferred from LolA to 249 LolB. Thus, the linker is not required for the transfer of RcsF from LolA to LolB. 250 251 Finally, we focused on the LolCDE ABC transporter in charge of extracting outer membrane 252 lipoproteins and transferring them to LolA. Over-expression (Extended Data Fig. 6a) of all 253 components of this complex failed to rescue normal targeting of RcsF∆19-47 to the outer 254 membrane (Extended Data Fig. 6b). Likewise, over-expressing the enzymes involved in 255 lipoprotein maturation (Lgt, LspA, and Lnt; Fig. 1) had no impact on membrane targeting 256 (Extended Data Fig. 7a, 7b). Thus, taken together, our results suggest that retention of RcsF∆19-257 47 in the inner membrane does not result from the impairment of a specific step, but rather from 258 less efficient processing of the truncated lipoprotein by the entire lipoprotein maturation 259 pathway (see Discussion). 260 13 Discussion 261 262 Lipoproteins are crucial for essential cellular processes such as envelope assembly and 263 virulence. However, despite their functional importance and their potential as targets for new 264 antibacterial therapies, we only have a vague understanding of the molecular factors that control 265 their biogenesis. By discovering the role played by N-terminal disordered linkers in lipoprotein 266 sorting, this study adds an important new layer to our comprehension of lipoprotein biogenesis 267 in Gram-negative bacteria. Critically, it also indicates that the current model of lipoprotein 268 sorting—that sorting between the two membranes is controlled by the 2 or 3 residues that are 269 adjacent to the lipidated cysteine40—needs to be revised. Lipoproteins with unstructured linkers 270 at their N-terminus are commonly found in Gram-negative bacteria including many pathogens 271 (see below); further work will be required to determine whether these linkers control lipoprotein 272 targeting in organisms other than E. coli, laying the foundation for designing new antibiotics. 273 274 It was previously shown that both lolA and lolB (but not lolCDE) can be deleted under specific 275 conditions21, suggesting at least one alternate route for the transport of lipoproteins across the 276 periplasm and their delivery to the outer membrane. During this investigation, we envisaged 277 the possibility that the linker could be required to transport lipoproteins via a yet-to-be-278 identified pathway independent of LolA/LolB. However, our observations that both RcsF and 279 RcsF∆19-47 were found in complex with LolA (Extended Data Fig. 5) and were transferred by 280 LolA to LolB (Fig. 4b) does not support this hypothesis. Instead, our data clearly indicate that 281 lipoproteins with N-terminal linkers still depend on the Lol system for extraction from the inner 282 membrane and transport to the outer membrane (Extended Data Fig. 1a); they also suggest 283 that N-terminal linkers improve lipoprotein processing by Lol (see below). 284 285 14 We note that two of the lipoproteins under investigation here, Pal and RcsF, have been reported 286 to be surface-exposed30,41,42. A topology model has been proposed to explain how RcsF reaches 287 the surface: the lipid moiety of RcsF is anchored in the outer leaflet of the outer membrane 288 while the N-terminal linker is exposed on the cell surface before being threaded through the 289 lumen of b-barrel proteins42. Thus, in this topology, the linker allows RcsF to cross the outer 290 membrane. It is therefore tempting to speculate that N-terminal disordered linkers may be used 291 by lipoproteins as a structural device to cross the outer membrane and reach the cell surface. It 292 is worth noting that N-terminal linkers are commonly found in lipoproteins expressed by the 293 pathogens Borrelia burgdorferi and Neisseria meningitides24,43,44; lipoprotein surface exposure 294 is common in these pathogens. In addition, the accumulation of RcsF∆19-47 in the inner 295 membrane (Fig. 2a) also suggests that Lol may be using N-terminal linkers to recognize 296 lipoproteins destined to the cell surface before their extraction from the inner membrane in 297 order to optimize their targeting to the machinery exporting them to their final destination 298 (BAM in the case of RcsF30,42,45). Investigating whether a dedicated Lol-dependent route exists 299 for surface-exposed lipoproteins will be the subject of future research. 300 301 Our work also delivers crucial insights into the functional importance of disordered segments 302 in proteins in general. Most proteins are thought to present portions that are intrinsically 303 disordered. For instance, it is estimated that 30-50% of eukaryotic proteins contain regions that 304 do not adopt a defined secondary structure in vitro46. However, demonstrating that these 305 unstructured regions are functionally important in vivo is challenging. By showing that an N-306 terminal disordered segment downstream of the Lol signal is required for the correct sorting of 307 lipoproteins, our work provides direct evidence that evolution has selected intrinsic disorder by 308 function. 309 310 15 In conclusion, the data reported here establish that the triage of lipoproteins between the inner 311 and outer membranes is not solely controlled by the Lol sorting signal; additional molecular 312 determinants, such as protein intrinsic disorder, are also involved. Our data further highlight 313 the previously unrecognized heterogeneity of the important lipoprotein family and call for a 314 careful evaluation of the maturation pathways of these lipoproteins. 315 316 DATA AVAILABILITY 317 All data generated or analysed during this study are included in this published article and its 318 supplementary information file. 319 320 REFERENCES 321 1. Silhavy, T.J., Kahne, D. & Walker, S. The bacterial cell envelope. 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Experiments in Molecular Genetics, (Cold Spring Harbor Laboratory Press, 456 New York, 1972). 457 54. Šali, A. & Blundell, T.L. Comparative Protein Modelling by Satisfaction of Spatial 458 Restraints. Journal of Molecular Biology 234, 779-815 (1993). 459 55. Pettersen, E.F. et al. UCSF Chimera - A visualization system for exploratory research 460 and analysis. Journal of Computational Chemistry 25, 1605-1612 (2004). 461 56. Guzman, L.M., Belin, D., Carson, M.J. & Beckwith, J. Tight regulation, modulation, and 462 high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 463 177, 4121-30 (1995). 464 465 466 ACKNOWLEDGMENTS 467 We thank Asma Boujtat for technical help. We are indebted to the members of the Collet 468 laboratory and to Nassos Typas (EMBL, Heidelberg) for helpful suggestions and discussions 469 and to Tom Silhavy (Princeton) for providing bacterial strains. J.S. was a research fellow of the 470 FRIA and J.F.C. is an Investigator of the FRFS-WELBIO. This work was funded by the 471 WELBIO, by grants from the F.R.S.-FNRS, from the Fédération Wallonie-Bruxelles (ARC 472 17/22-087), from the European Commission via the International Training Network 473 Train2Target (721484), and from the EOS Excellence in Research Program of the FWO and 474 FRS-FNRS (G0G0818N). 475 476 AUTHOR CONTRIBUTIONS 477 19 J.-F.C., J.E.R., J.S., and S.H.C. designed and performed the experiments. J.E.R., J.S., and 478 S.H.C. constructed the strains and cloned the constructs. J.-F.C., J.E.R., J.S., S.H.C., and A.M. 479 analyzed and interpreted the data. B.I.I. performed the structural analysis. J.-F.C., J.E.R., and 480 J.S. wrote the manuscript. All authors discussed the results and commented on the manuscript. 481 20 FIGURE LEGENDS 482 483 Figure 1. Structural analysis of lipoproteins reveals that half of outer membrane 484 lipoproteins display an intrinsically disordered linker at the N-terminus. 485 Structures were generated via comparative modeling (Methods). X-ray and cryo-EM structures 486 are green, NMR structures are cyan, and structures built via comparative modeling from the 487 closest analog in the same PFAM group are orange. In all cases, the N-terminal linker is 488 magenta. Lipoproteins targeting the outer membrane: Pal, OsmE, NlpE, NlpC, MltB, NlpI, 489 MltC, RcsF, YajI, YcfL, YbaY, RlpA, NlpD, YcaL. The 29 remaining lipoproteins are shown 490 in Extended Data Figure 2. 491 492 Figure 2. The N-terminal linker displayed by lipoproteins is important for outer 493 membrane targeting. 494 a, b, c. The outer membrane (OM) and inner membrane (IM) were separated via centrifugation 495 in a three-step sucrose density gradient (Methods). While (c) RcsFWT, (d) NlpDWT, and (e) 496 PalWT were found predominantly in the OM, RcsF∆19-47, NlpD∆29-64, and Pal∆26-56 were 497 substantially retained in the IM. Data are presented as the ratio of signal intensity in a single 498 fraction to the total intensity in all fractions. All variants were expressed from plasmids 499 (Extended Data Table 4). DsbD and Lpp were used as controls for the OM and IM, 500 respectively. d. The Rcs system is constitutively active when RcsF’s linker is missing. Rcs 501 activity was measured with a beta-galactosidase assay in a strain harboring a transcriptional 502 rprA::lacZ fusion (Methods). Results were normalized to expression levels of RcsF variants 503 (mean ± standard deviation; n = 6 biologically independent experiments) e. Phase-contrast 504 images of the envC::kan ∆nlpD mutant complemented with NlpDWT or NlpD∆29-64. NlpD∆29-64 505 only partially rescues the chaining phenotype of the envC::kan ∆nlpD double mutant. Scale 506 21 bar, 5 µm. f. Expression of Pal∆26-56 does not rescue the sensitivity of the pal::kan mutant to 507 SDS-EDTA. Cells were grown in LB medium at 37 °C until OD600 = 0.5. Tenfold serial 508 dilutions were made in LB, plated onto LB agar or LB agar supplemented with 0.01% SDS and 509 0.5 mM EDTA, and incubated at 37 °C. Images in a, b, c, e, and f are representative of 510 biological triplicates. Graphs in a, b, and c were created by spline analysis of curves 511 representing a mean of three independent experiments. 512 513 Figure 3. The length and the disordered character of the RcsF linker play key roles in 514 RcsF targeting to the outer membrane. 515 a. The outer membrane (OM) and inner membrane (IM) were separated via centrifugation in a 516 three-step sucrose density gradient (Methods). DsbD and Lpp were used as controls for the OM 517 and IM, respectively. The longer the linker, the more protein was correctly translocated to the 518 IM. Bar graphs denote mean ± standard deviation of n = 3 biologically independent 519 experiments. Images are representative of experiments and immunoblots performed in 520 biological triplicate. b. Rcs activity was measured with a beta-galactosidase assay in a strain 521 harboring a transcriptional rprA::lacZ fusion (Methods). Results were normalized to expression 522 levels of RcsF variants (mean ± standard deviation of n = 6 biologically independent 523 experiments). Rcs activity relates to the quantity of RcsF retained in the inner membrane. c. 524 RcsF mutants harboring alpha helical linkers (RcsFFkpA and RcsFcol) were subjected to two 525 consecutive centrifugations in sucrose density gradients (Methods). Both mutants were 526 inefficiently translocated from the IM to the OM (mean ± standard deviation of n = 3 527 biologically independent experiments). Images are representative of experiments and 528 immunoblots performed in biological triplicate. d. The Rcs system was constitutively active in 529 RcsFFkpA and RcsFcol strains; activation levels were comparable to those of RcsF∆19-47. Rcs 530 activity was measured as in b. Results were normalized as in b. 531 22 532 Figure 4. N-terminal disordered linkers interact with the Lol system to target lipoproteins 533 to the outer membrane. 534 a. Deleting Lpp rescues normal targeting of RcsF∆19-47 and NlpD∆29-64 to the outer membrane. 535 The outer and inner membranes were separated via centrifugation in a sucrose density gradient 536 (Methods). Whereas RcsF∆19-47 and NlpD∆29-64 accumulate in the inner membrane of cells 537 expressing Lpp, the most abundant Lol substrate, they are normally targeted to the outer 538 membrane in cells lacking Lpp (mean ± standard deviation of n = 3 biologically independent 539 experiments). b. In vitro pull-down experiments show that RcsFWT and RcsF∆19-47 are 540 transferred from LolA to LolB. LolA-RcsFWT and LolA- RcsF∆19-47 complexes were obtained 541 by LolA-His affinity chromatography followed by size exclusion chromatography (Methods). 542 Each complex was incubated with LolB-Strep that was previously purified via Strep-Tactin 543 affinity chromatography (Methods). Both RcsF variants were eluted in complex with LolB-544 strep, while LolA was only present in the flow through. I, input; FT, flow through; E, eluate. 545 546 23 FIGURES 547 Figure 1 548 549 550 24 551 Figure 2 552 553 25 554 Figure 3 555 556 557 26 558 Figure 4 559 560 561 562 563 564 565 566 27 567 METHODS 568 569 Bacterial growth conditions 570 Bacterial strains used in this study are listed in Extended Data Table 3. Bacterial cells were 571 cultured in Luria broth (LB) at 37 °C unless stated otherwise. The following antibiotics were 572 added when appropriate: spectinomycin (100 µg/mL), ampicillin (200 µg/mL), 573 chloramphenicol (25 µg/mL), and kanamycin (50 µg/mL). L-arabinose (0.2%) and isopropyl-574 β-D-thiogalactoside (IPTG) were used for induction when appropriate. 575 576 Bacterial strains and plasmids 577 DH300 (a derivative of Escherichia coli MG1655 carrying a chromosomal rprA::lacZ fusion at 578 the λ attachment site47) was used as wildtype throughout the study. All deletion mutants were 579 obtained by transferring the corresponding alleles from the Keio collection48 (kanR) into 580 DH30047 via P1 phage transduction. Deletions were verified by PCR and the absence of the 581 protein was verified via immunoblotting (when possible). If necessary, the kanamycin cassette 582 was removed via site-specific recombination mediated by the yeast Flp recombinase with 583 pCP20 vector49. All strains expressing the RcsF mutants used for subcellular fractionation 584 lacked rcsB in order to prevent induction of Rcs. 585 586 The plasmids used in this study are listed in Extended Data Table 4 and the primers appear in 587 Extended Data Table 5. RcsF, Pal, and NlpD were expressed from the low-copy vector 588 pAM23850 containing the SC101 origin of replication and the lac promoter. To produce pSC202 589 for RcsF expression, rcsF (including approximately 30 base pairs upstream of the coding 590 sequence) was amplified by PCR from the chromosome of DH300 (primer pair SH_RcsF(PstI)-591 28 R and SH_RcsFU-R (kpnI)-F). The amplification product was digested with KpnI and PstI and 592 inserted into pAM238, resulting in pSC202. nlpD was amplified using primers JR1 and JR2 593 and pal was amplified with primers JS145 and JS146. Amplification products were digested 594 with PstI-XbaI and KpnI-XbaI, respectively, generating pJR8 (for NlpD expression) and pJS20 595 (for Pal expression). To clone rcsFΔ19-47, the nucleotides encoding the RcsF signal sequence 596 were amplified using primers SH_RcsFUR(kpnI)_F and SH_RcsFss-Fsg (NcoI)_R, and those 597 encoding the RcsF signaling domain were amplified using primers SH_RcsFss-Fsg (NcoI)_R 598 and SH_RcsF(PstI)_R. In both cases, pSC202 was used as template. Then, overlapping PCR 599 was performed using SH_RcsFUR(kpnI)_F and SH_RcsF(PstI)_R from the two PCR products 600 previously obtained. The final product was digested with KpnI and PstI, and ligated with 601 pAM238 pre-digested with the same enzymes, yielding pSC201. To add a GS linker (Ser-Gly-602 Ser-Gly-Ser-Gly-Ala-Met) into pSC201, the primers SH_GS linker_F and SH_GS linker_R 603 were mixed, boiled, annealed at room temperature, and ligated with pSC201 pre-digested with 604 NcoI, generating pSC198. pSC199 was generated similarly, but using primers SH_SG linker_F 605 and SH_SG linker_R and plasmid pSC198. pSC200 was generated using primers SH_Da 606 linker_F and SH_SG linker_R and plasmid pSC199. The pal allele lacking the linker region 607 (palΔ26-56) was created via overlapping PCR. The pJS20 plasmid served as template for PCR 608 with the M13R/M13F external primers and JS152/JS153 internal primers. The truncated allele 609 was cloned into pAM238 at the same restriction sites as the full-length allele, producing pJS24. 610 The nlpD allele lacking the linker regions (nlpDΔ29-64) was created via overlapping PCR. E. coli 611 chromosomal nlpD served as template for the PCR, with JR1/JR2 as external primers and 612 JR7/JR8 as internal primers. The truncated allele was then cloned into pAM238 at the same 613 restriction sites as the full-length allele, producing pJR10. 614 615 29 rcsFFkpA and rcsFcol were obtained by inserting DNA sequences corresponding to helical linker 616 fragments (FkpA Ser94-Glu125 and colicin IA Ile213-Lys282) into rcsFΔ19-47 at NcoI and RsrII 617 restriction sites. The fkpA gene fragment was amplified from the E. coli MC4100 chromosome 618 (JS50/JS51 primers) and the cia gene fragment was chemically synthetized as a gene block by 619 Integrated DNA Technologies (IDT). The resulting plasmids were pJS18 and pJS27, 620 respectively. pAM238 does not contain the lacIq repressor. Therefore, to enable expression-621 level regulation by IPTG, strains containing the pAM238 plasmids expressing RcsF variants 622 were co-transformed with pET22b, a high-copy plasmid from a different incompatibility group 623 (pBR223 origin of replication; Novagen) containing the lacIq repressor. Chromosomal 624 insertion of RcsFΔ19-47 was performed via λ-Red recombineering51 with pSIM5-Tet plasmid (a 625 gift of D. Hughes). In the first step, the cat-sacB cassette was introduced and later replaced by 626 mutant rcsF. 627 628 The chromosomal lolCDE operon was amplified via PCR using primers JS277 and JS278 629 (adding a C-terminal His-tag to LolE) and then inserted into pBAD33 using the restriction sites 630 PstI and XbaI, resulting in pJR203. The expression level of LolE-His was verified via 631 immunoblotting. 632 633 The sequence encoding lolB without its N-terminal cysteine was first amplified from the 634 chromosome via PCR using primers JR50/PL387 (adding a C-terminal Strep-tag). It was then 635 cloned into pET28a using the restriction sites XbaI and PstI. lolA was amplified using 636 chromosomal lolA as PCR template for primers JR30/JR31 (JR31 contains the sequence of a 637 His-tag) and then cloned into pBAD18 using KpnI and XbaI, resulting in pJR48. 638 639 30 The genes encoding Lgt and Lnt were amplified from the chromosome with PCR primers 640 AG389/AG403 and AG393/JR74, respectively. AG403 and JR74 also encode a Myc-tag. PCR 641 products were cloned into pAM238 using KpnI and PstI. Expression levels were verified via 642 immunoblotting (data not shown). lspA was amplified with PCR primers JR77/JR78. The PCR 643 product was cloned into pSC213, a modified pAM238 with a ribosome binding site and a C-644 terminal Flag tag, using NcoI and BamHI. Expression of LspA-Flag was induced by adding 25 645 µM IPTG. Expression levels were verified with immunoblots (data not shown). 646 647 Cell fractionation and sucrose density gradients 648 Cell fractionation was performed as described previously52 with some modifications. Four 649 hundred milliliters of cells were grown until the optical density at 600 nm (OD600) of the culture 650 reached 0.7. Cells were harvested via centrifugation at 6,000 x g at 4 °C for 15 min, washed 651 with TE buffer (50 mM Tris-HCl pH 8, 1 mM EDTA), and resuspended in 20 mL of the same 652 buffer. The washing step was skipped with the Dlpp strains to prevent the loss of outer 653 membrane vesicles. DNase I (1 mg; Roche), 1 mg RNase A (Thermo Scientific), and a half 654 tablet of a protease inhibitor cocktail (cOmplete EDTA-free Protease Inhibitor Cocktail tablets; 655 Roche) were added to cell suspensions, and cells were passed through a French pressure cell at 656 1,500 psi. After adding MgCl2 to a final concentration of 2 mM, the lysate was centrifuged at 657 5,000 x g at 4 °C for 15 min in order to remove cell debris. Then, 16 mL of supernatant were 658 placed on top of a two-step sucrose gradient (2.3 mL of 2.02 M sucrose in 10 mM HEPES pH 659 7.5 and 6.6 mL of 0.77 M sucrose in 10 mM HEPES pH 7.5). The samples were centrifuged at 660 180,000 x g for 3 h at 4 °C in a 55.2 Ti Beckman rotor. After centrifugation, the soluble fraction 661 and the membrane fraction were collected. The membrane fraction was diluted four times with 662 10 mM HEPES pH 7.5. To separate the inner and the outer membranes, 7 mL of the diluted 663 membrane fraction were loaded on top of a second sucrose gradient (10.5 mL of 2.02 M sucrose, 664 31 12.5 mL of 1.44 M sucrose, 7 mL of 0.77 M sucrose, always in 10 mM HEPES pH 7.5). The 665 samples were then centrifuged at 112,000 x g for 16 h at 10 °C in a SW 28 Beckman rotor. 666 Approximately 30 fractions of 1.5 mL were collected and odd-numbered fractions were 667 subjected to SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with specific 668 antibodies. Graphs were created in GraphPad Prism 9 via spline analysis of the curves 669 representing a mean of three independent experiments. 670 671 Immunoblotting 672 Protein samples were separated via 10% or 4-12% SDS-PAGE (Life Technologies) and 673 transferred onto nitrocellulose membranes (GE Healthcare Life Sciences). The membranes 674 were blocked with 5% skim milk in 50 mM Tris-HCl pH 7.6, 0.15 M NaCl, and 0.1% Tween 675 20 (TBS-T). TBS-T was used in all subsequent immunoblotting steps. The primary antibodies 676 were diluted 5,000 to 20,000 times in 1% skim milk in TBS-T and incubated with the membrane 677 for 1 h at room temperature. The anti-RcsF, anti-DsbD, anti-Lpp, anti-NlpD, anti-LolA, and 678 anti-LolB antisera were generated by our lab. Anti-Pal was a gift from R. Lloubès, and anti-His 679 is a peroxidase-conjugated antibody (Qiagen). The membranes were incubated for 1 h at room 680 temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) at a 1:10,000 681 dilution. Labelled proteins were detected via enhanced chemiluminescence (Pierce ECL 682 Western Blotting Substrate, Thermo Scientific) and visualized using X-ray film (Fuji) or a 683 camera (Image Quant LAS 4000 and Vilber Fusion solo S). In order to quantify proteins levels, 684 band intensities were measured using ImageJ version 1.46r (National Institutes of Health). 685 686 β-galactosidase assay 687 β-galactosidase activity was measured as described previously53. Graphs representing a mean 688 of six experiments with standard deviation were prepared in GraphPad Prism. Expression-level 689 32 estimations were performed as follows. Cultures used for β-galactosidase activity (0.5 mL per 690 culture) were precipitated with 10% trichloroacetic acid, washed with ice-cold acetone, and 691 resuspended in 0.2 mL Laemmli SDS sample buffer. Samples (5 µL) were subjected to SDS-692 PAGE and immunoblotted with anti-RcsF antibody. 693 694 SDS-EDTA sensitivity assay 695 Cells were grown in LB at 37 °C until they reached an OD600 of 0.7. Tenfold serial dilutions 696 were made in LB and plated on LB agar supplemented with spectinomycin (100 µg/mL) when 697 necessary. Plates were incubated at 37 °C. To evaluate the sensitivity of the pal mutant, plates 698 were supplemented with 0.01% SDS and 0.5 mM EDTA. 699 700 Microscopy image acquisition 701 Cells were grown in LB at 37 °C until OD600 = 0.5. Cells growing in exponential phase were 702 spotted onto a 1% agarose phosphate-buffered saline pad for imaging. Cells were imaged on a 703 Nikon Eclipse Ti2-E inverted fluorescence microscope with a CFI Plan Apochromat DM 704 Lambda 100X Oil, N.A. 1.45, W.D. 0.13 mm objective. Images were collected on a Prime 95B 705 25 mm camera (Photometrics). We used a Cy5-4050C (32 mm) filter cube (Nikon). Image 706 acquisition was performed with NIS-Element Advance Research version 4.5. 707 708 Protein purification 709 JR90 cells were grown in LB supplemented with kanamycin (50 µg/mL) at 37 °C. When the 710 culture OD600 = 0.5, the expression of cytoplasmic LolB-Strep was induced with 1 mM IPTG. 711 Cells (1 L) were pelleted when they reached OD600 = 3 and resuspended in 25 mL of buffer A 712 (200 mM NaCl and 50 mM NaPi, pH 8) containing one tablet of cOmplete EDTA-free Protease 713 Inhibitor Cocktail (Roche). Cells were lysed via two passages through a French pressure cell at 714 33 1,500 psi. The lysate was centrifuged at 30,000 x g for 40 min at 4 °C in a JA 20 rotor and the 715 supernatant was mixed with Strep-Tactin resin (IBA Lifesciences) previously equilibrated with 716 buffer A. After washing the resin with 10 column volumes of buffer A, LolB-Strep was eluted 717 with 5 column volumes of buffer A supplemented with 5 mM desthiobiotin. LolB-Strep was 718 finally desalted using a PD10 column (GE Healthcare). 719 720 Soluble LolA-RcsFWT and LoA-RcsFΔ19-47 complexes were purified via affinity 721 chromatography as follows. Cells co-expressing LolA either with wild-type RcsF (JR47) or 722 RcsFΔ19-47 (JR44) were grown in LB at 37 °C supplemented with 200 µg/mL ampicillin until 723 OD600 = 0.5. Protein expression was then induced with 0.2% arabinose. Cells (1 L) were 724 pelleted at OD600 = 3 and resuspended in 25 mL of buffer A containing one tablet of protease 725 inhibitor cocktail. Cells were lysed via two passages through a French pressure cell at 1,500 726 psi. The lysate was centrifuged at 45,000 x g for 30 min at 4 °C using a 55.2 Ti Beckman rotor. 727 To obtain the soluble fraction, the supernatant was centrifuged at 180,000 x g for 1 h at 4 °C 728 using the same rotor. The supernatant was added to a His Trap HP column (Merck) previously 729 equilibrated with buffer A. The column was washed with 10 column volumes of buffer A 730 supplemented with 20 mM imidazole and LolA-His was eluted using a gradient of imidazole 731 (from 20 mM to 300 mM). The fractions obtained were analyzed via SDS-PAGE; LolA was 732 detected around 25 kDa (data not shown). RcsF variants were detected via immunoblotting with 733 an anti-RcsF antibody. Fractions containing LolA-RcsF variants were pooled, concentrated to 734 1 mL using a Vivaspin 4 Turbo concentrator (Cut-off 5 kDa; Sartorius), and purified via size-735 exclusion chromatography with a Superdex S75-10/300 column (GE Healthcare). 736 737 Pull down and transfer of RcsF variants from LolA to LolB 738 34 LolB-Strep was incubated at 30 °C for 20 min under agitation with LolA-RcsFWT or with LolA-739 RcsFΔ19-47 (LolA-RcsFWT and LolA-RcsFΔ19-47 complexes were purified as described above). 740 The mixture was added to magnetic Strep beads (MagStrep type 3 beads, IBA Life science) 741 previously equilibrated with buffer A and incubated for 30 min at 4 °C on a roller. After washing 742 the beads with the same buffer, LolB-Strep was eluted with buffer A supplemented with 50 mM 743 biotin. Samples were analyzed via SDS-PAGE and LolA and LolB were detected with 744 Coomassie Brilliant Blue (Bio-Rad). RcsF was detected via immunoblotting with an anti-RcsF 745 antibody. 746 747 Structural analysis of lipoproteins 748 When X-ray, cryo-EM, or NMR structures were available, the missing residues were completed 749 through comparative modeling using MODELLER version 9.2254. If no structure of the 750 lipoprotein was available, then the most pertinent analogous structure from proteins belonging 751 to the same PFAM group was used as template for comparative modeling. The linker was 752 defined as the unstructured fragment from the N-terminal Cys of the mature form until the first 753 residue with well-defined secondary structure (α-helix or β-strand) belonging to a globular 754 domain. Short, intermediate, and long linkers had lengths of <12, 12-22, and >22 residues, 755 respectively. Images were generated using UCSF Chimera version 1.13.155. 756 757 35 LEGENDS FOR FIGURES IN THE EXTENDED DATA 758 759 Extended Data Figure 1. Lipoprotein maturation and sorting in the E. coli cell envelope. 760 a. After processing by Lgt (step 1), LspA (step 2), and Lnt (step 3), a new lipoprotein either 761 remains in the inner membrane or is extracted by the LolCDE complex (step 4), depending on 762 the residues at position +2 and +3. LolCDE transfers the lipoprotein to the periplasmic 763 chaperone LolA (step 5), which delivers the lipoprotein to LolB (step 6). LolB, a lipoprotein 764 itself, inserts the lipoprotein in the outer membrane using a poorly understood mechanism (step 765 7). b. Schematic of lipoprotein structural domains. The N-terminal signal sequence targets the 766 lipoprotein to the cell envelope; the last four amino acid residues of the signal sequence form 767 the lipobox. The last residue of the lipobox is the invariant cysteine that undergoes lipidation. 768 This cysteine, which is the first residue of the mature lipoprotein, is directly followed by the 769 sorting signal, a sequence of 2 or 3 amino acids that controls the sorting of mature lipoproteins 770 between the inner and outer membranes. The C-terminal portion of a mature lipoprotein is a 771 globular domain. An intrinsically disordered linker separates the sorting signal from the 772 globular domain in about half of E. coli lipoproteins (Fig. 1; Extended Data Fig. 2; Extended 773 Data Table 1). The lengths of the deleted disordered linkers of the unrelated lipoproteins RcsF, 774 Pal, and NlpD are indicated. LP, lipoprotein. 775 776 Extended Data Figure 2. Structural analysis of lipoproteins reveals that half of outer 777 membrane lipoproteins display an intrinsically disordered linker at the N-terminus. 778 Structures were generated via comparative modeling. X-ray and cryo-EM structures are green, 779 NMR structures are cyan, and structures built via comparative modeling from the closest analog 780 in the same PFAM group are orange. In all cases, the N-terminal linker is magenta. Lipoproteins 781 targeting the outer membrane: AmiD, BamB, BamC, HslJ, MltA, LoiP, LpoB, Blc, BamE, 782 CsgG, EmtA, GfcE, BamD, LpoA, LolB, LptE, MlaA, MliC, YddW, YedD, YghG, YfeY, 783 36 YbjP, YiaD, YbhC, PqiC, YgeR, YfiB, YraP. Lipoproteins targeting the IM: DcrB, MetQ, 784 NlpA, YcjN, YehR, ApbE. Synthetic constructs: RcsFGS, RcsFGS2, RcsFGS3, RcsF∆19-47, 785 RcsFFkpA, RcsFcol, NlpD∆29-64, Pal∆26-56. 786 787 Extended Data Figure 3. Expression levels of RcsF∆19-47, Pal∆26-56, and NlpD∆29-64. 788 Cells were grown at 37 °C in LB until OD600 = 0.5 and precipitated with trichloroacetic acid 789 (Methods). Immunoblots were performed with a-RcsF, a-NlpD, and a-Pal antibodies 790 (Methods). All images are representative of three independent experiments. 791 792 Extended Data Figure 4. Schematic of RcsF variants used in this study and their 793 distributions in the outer membrane (OM) and inner membrane (IM). 794 RcsFGS, RcsFGS2, and RcsFGS3 have linkers that are disordered and mostly consist of GS repeats. 795 The linker of RcsFGS is the same length as the linker of RcsFWT. RcsFGS2 and RcsFGS3 are shorter 796 than RcsFWT. Regions of RcsFFkpA and RcsFcol fold into alpha helices borrowed from the 797 sequences of FkpA and colicin Ia, respectively. 798 799 Extended Data Figure 5. Complexes between LolA and RcsFWT or RcsF∆19-47 can be 800 purified. 801 Both RcsFWT (a) and RcsF∆19-47 (b) were eluted in complex with LolA-His via affinity 802 chromatography followed by size exclusion chromatography. Gel filtration was performed with 803 a Superdex S75-10/300 column. Samples were analyzed via SDS-PAGE and proteins, 804 including LolA-His, were stained with Coomassie Brilliant Blue (Methods). RcsF variants were 805 detected by immunoblotting fractions with a-RcsF antibodies. Images are representative of 806 three independent experiments. 807 808 37 Extended Data Figure 6. Overexpression of Lol CDE does not restore targeting of RcsF∆19-809 47. 810 a. Expression level of LolCDE-His. Cells were grown in LB plus 0.2% arabinose at 37 °C until 811 OD600 = 0.7 (Methods). Membrane and soluble fractions were separated with a sucrose density 812 gradient (Methods). LolE-His was detected in the membrane fraction by immunoblotting with 813 a-His (Methods). Images are representative of three independent experiments. b. The outer 814 membrane (OM) and inner membrane (IM) were separated with a sucrose density gradient. 815 Expression of LolCDE did not rescue OM targeting of RcsF∆19-47. Images are representative of 816 experiments performed in biological triplicate. 817 818 Extended Data Figure 7. Overexpressing Lgt, LspA, and Lnt does not rescue the targeting 819 of RcsF∆19-47 to the outer membrane. 820 a. Expression levels of Lgt, LspA, and Lnt. Cells were grown in LB (plus 25 µM IPTG for cells 821 expressing LspA) at 37 °C until OD600 = 0.7 (Methods). Outer membrane (OM) and inner 822 membrane (IM) were separated with a sucrose density gradient (Methods). Lgt-Myc and Lnt-823 Myc were detected in the IM via immunoblotting with a-Myc. LspA-Flag was detected in the 824 IM with a-Flag. b. Cells overexpressing Lgt, LspA, or Lnt were exposed to a sucrose density 825 gradient (Methods). RcsF∆19-47 was retained in the IM in all conditions. Images are 826 representative of three independent experiments. 827 828 38 EXTENDED DATA FIGURES 829 Extended Data Figure 1 830 831 832 833 39 Extended Data Figure 2 834 835 836 40 Extended Data Figure 3 837 838 839 41 Extended Data Figure 4 840 841 842 42 Extended Data Figure 5 843 844 43 Extended Data Figure 6 845 846 44 Extended Data Figure 7 847 848 45 EXTENDED DATA TABLES 849 850 Extended Data Table 1: List of the verified lipoproteins of E. coli used for the structural 851 analysis in this study. 852 Attached Excel sheet 853 854 Extended Data Table 2: RcsF mutants used in this study and the amino acid sequences of 855 their corresponding N-terminal linkers. The acylated cysteine is the first residue listed. 856 RcsF linkers Amino acid sequence RcsFWT CSMLSRSPVEPVQSTAPQPKAEPAKPKAPRATPV RcsFΔ19-47 CSMGPV RcsFGS CSMSLFDAPAMSGSGSGAMSGSGSGAMPV RcsFGS2 CSMSGSGSGAMSGSGSGAMPV RcsFGS3 CSMSGSGSGAMPV RcsFFkpA CSMGSDQEIEQTLQAFEARVKSSAQAKMEKDAADNEPV RcsFcol CSMGILDTRLSELEKNGGAALAVLDAQQARLLGQQTRNDRAISEARNKL SSVTESLNTARNALTRAEQQLTQQKPV 857 858 859 46 Extended Data Table 3: E. coli strains used in this study. 860 Strains Genotype and description Source DH300 rprA-lacZ MG1655 (argF-lac) U169 47 Keio collection single mutants rcsF::kan, rcsB::kan, pal::kan, nlpD::kan, envC::kan 48 XL1-Blue endA1 gyrA96 (nalR) thi-1 recA1 relA1 lac glnV44F’ [::Tn10 proAB+ lacIq D(lacZ)M15] hsdR17 (rK- mK+) Stratagene BL21 F- ompT hsdSB (rB- mB-) gal dcm (DE3) Novagen JS41 DH300 DrcsF pAM238 This study JS265 DH300 DrcsF pJS18 This study JS346 DH300 DrcsF rcsB::kan pET22b This study JS267 JS346 pJS18 This study JS325 DH300 pal::kan This study JS331 JS325 pJS20 This study JS345 JS325 pJS24 This study JS360 DH300 DrcsF pJS27 This study JS363 JS346 pJS27 This study JS364 DH300 DrcsF pSC202 This study JS372 DH300 DrcsF pSC201 This study JS395 JS346 pSC198 This study JS396 JS346 pSC199 This study JS397 JS346 pSC200 This study JS398 JS346 pSC201 This study JS573 JS346 pSC202 This study JS574 DH300 DrcsF pSC198 This study JS575 DH300 DrcsF pSC199 This study 47 JS576 DH300 DrcsF pSC200 This study JS639 DrcsB lpp::kan rcsF::rcsFD19-47 This study JR30 nlpD::kan This study JR31 JR30 pJR8 This study JR32 JR30 pJR10 This study JR2 DH300 pAM238 This study JR88 BL21 rcsF::kan This study JR90 JR88 pET28-cytoplasmic LolB-Strep This study JR187 rcsB::kan rcsF::rcsFD19-47 This study JR149 DnlpD This study JR121 DnlpD envC::kan This study JR122 JR121 pJR8 This study JR123 JR121 pJR10 This study JR188 JR187 pAM238 This study JR191 JR187 pAG833 This study JR204 JR187 pJR203 This study JR194 JR187 pBAD33 This study JR211 JR187 pJR209 This study JR257 JR187 pJR239 This study JR274 JR149 lpp::kan This study JR279 JR274 pJR10 This study JR292 JS325 pAM238 This study JR293 JR187 pSC213 This study JR44 rcsB::kan rcsF::rcsFD19-47 pJR48 This study 48 JR47 rcsB::kan pJR48 This study JR77 rcsB::kan rcsF::rcsFD19-47 pBAD18 This study JR78 rcsB::kan pBAD18 This study 861 862 49 Extended Data Table 4: Plasmids used in this study. 863 Plasmids Features Source pAM238 IPTG-regulated Plac, pSC101-based, spectinomycin (no lacIQ) 50 pBAD18 Arabinose inducible PBAD, ampicillin 56 pBAD33 Arabinose inducible PBAD, chloramphenicol 56 pET28a IPTG regulated T7 promoter, kanamycin Novagen pET22b IPTG regulated T7 promoter, ampicillin Novagen pCP20 FLP+, l cI857+, l PR Repts, ampicillin, chloramphenicol 49 pSIM5-Tet pSC101 plasmid, repAts, tetRA, l-Red (Gram-Beta-Exo), cI857, tetracycline Gift from D. Hughes pJS18 pAM238 RcsFFKpA FkpA linker (S94-E125) This study pJS20 pAM238 PalWT This study pJS24 pAM238 PalD26-56 This study pJS27 pAM238 RcsFcol Colicin Ia linker (I213-K282) This study pSC198 pAM238 RcsFGS3 (C16S17M18S19GSGSGAMG) This study pSC199 pAM238 RcsFGS2 (C16S17M18S19GSGSGAMSGSGSGAM G) This study pSC200 pAM238 RcsFGS (C16S17M18S19LFDAPAMSGSGSGAM SGSGSGAMG) This study pSC201 pAM238 RcsFD19-47 (C16S17M18G19P20) This study pSC202 pAM238 RcsFWT This study pJR8 pAM238 NlpDWT This study pJR10 pAM238 NlpDD29-64 (C26S27D28A29) This study pJR48 pBAD18 LolA-6xHis This study pJR90 pET28 Cytoplasmic LolB-Strep This study 50 pJR203 pBAD33 LolCDE-6xHis This study pJR209 pAM238 Lnt-Myc This study pJR239 pSC213 LspA-Flag This study pSC213 pAM238, IPTG-regulated Plac , lacIQ, triple Flag tag This study pAG833 pAM238 Lgt-Myc This study 864 865 866 867 Extended Data Table 5: Primers used in this study. 868 Primer Sequence 5’ to 3’ JS50_FkpAlinker _fw acatccatggggtccgaccaagagatcgaac JS51_FkpAlinker _rv atgtcggaccggttcgttatcagccgcgtc JS143_Pal_-100b cgtcttccggcaactgatgg JS144_Pal_+100b ttggtgcctgagcaaaagcg JS145_Pal_fw ACATggtaccTTAATTGAATAGTAAAGGAATC JS146_Pal_rv ATGTtctagaTTAgtaaaccagtaccgcac JS152_PalNoLink er_overlapPCR_ fw tgttcttccaacCAGGCTCGTCTGCAAATG JS153_PalNoLink er_overlapPCR_ rv CAGACGAGCCTGgttggaagaacatgccgc JS277_LolCDEHi s_fw ACATtctagaTCTTTGCTACAGCAACCAGAC JS278_LolCDE_ His_rv ATGTctgcagTTAGTGATGGTGATGGTGATGACCctggccgctaaggactcg JS289_lred_catSa cBin_RcsF_fw tcctgattcaatattgacgttttgatcatacattgaggaaatactAAAATGAGACGTTGATCGG CACG 51 JS290_lred_catSa cBin_RcsF_rev tatagggcgagcgaataacgcctatttgctcgaactggaaactgcATCAAAGGGAAAACTGT CCA JS291_lred_RcsF _catSacBout_fw tcctgattcaatattgacgttttgatcatacattgaggaaatactATGCGTGCTTTACCGATCTG TT JS292_lred_RcsF _catSacBout_rv tatagggcgagcgaataacgcctatttgctcgaactggaaactgcTCATTTCGCCGTAATGTT AAGC JS293_junction1lr ed_RcsFup_fw gcggagctgttaaaggctg JS294_junction2lr ed_RcsFdown_rv gagcaatgagatgcagttcg JS295_junction1lr ed_cat-out_rv CGGGCAAGAATGTGAATAAAGG JS296_junction2lr ed_sacB-out_fw GCTGTACCTCAAGCGAAAGG M13R CAGGAAACAGCTATGACCATG M13F TGTAAAACGACGGCCAGT PL145_rcsF_- 100b cgctttttaccagacctggc PL146_rcsF_+10 0 atatcattcaggacgggcgcttgccc PL153_rcsB_- 100b acatctgattcgtgagaagg PL154_rcsB+100 b taatgggaatcgtaggccgg PL168_Fw_lpp_- 100 CAATTTTTTTATCTAAAACCCAGCG PL169_Rv_lpp_+ 100 CCAGAGCAAGGGAATATGTTACGCG SH_Da linker_F CATGaGcTTATTCGACGCGCCGGc SH_Da linker_R catggCCGGCGCGTCGAATAAgCt SH_RcsF(PstI)_R gagaCTGCAGtcaTTTCGCCGTAATGTTAAG SH_RcsFUR(kpn I)_F GAGGGTACCcgttttgatcatacattg RcsFss-Fsg (NcoI)_F GCGGCTGTTCCATGGggccggtccgaatttatac RcsFss-Fsg (NcoI)_R ggaccggccCCATGGAACAGCCGCTTAGCATGAG SH_GS linker_F CATGagtggctctggatctggtgc 52 SH_GS linker_R catggcaccagatccagagccact JR1_NlpD_fw GAGATCTAGATTATTAACCAATTTTTCCTGGGGGATAA JR2_NlpD_rv AGAGCTGCAGTTATCGCTGCGGCAAATAACGCA JR7_NlpDoverlap _fw GGCTGGCAGGCTGTTCTGACGCGCAGCAACCGCAAATTCA JR8_NlpDoverlap _rv TGAATTTGCGGTTGCTGCGCGTCAGAACAGCCTGCCAGCC JR23_Fw_NlpD- 98 CAGGTCAGCGTATCGTGAACATC JR24_Rv_NlpD+ 100 TCATTTAAATCATGAACTTTCAGCG JR30_Fw_LolA_- 28_pBAD18 ACATGGTACCCGGGAGTGACGTAATTTGAGGAAT JR31_Rev_LolA_ His_pBAD18 ATGTTCTAGAttaatgatgatgatgatgatgctcgaGCTTACGTTGATCATCTACC GTGAC JR50_Rev_cytopl asmic_LolB_nost op_StrepTag_stop CCAACTCGAGTCACTTTTCGAACTGCGGGTGGCTCCAGCTTGCTTT CACTATCCAGTTATCCAT JR56-Fw--100- envC GTTGTCGCTG ATGGGTA JR57-Rev- +100envC AATCATCAATGACGATGGCA JR74-Rev-Lnt- myctag-PstI AAAAACTGCAGctacaggtcttcttcgctaatcagtttctgttcgcttgcTTTACGTCGCTG ACGCAGAC JR77-Fw-NcoI- LspA gagaCCATGGgtAGTCAATCGATCTGTTCAAC JR78-Rev-LspA- no stop-BamHI gagaGGATCCTTGTTTTTTCGCTCTAG AG389_lgt_- 49_Fw_KpnI AAAAAggtaccTTCAATCGCTGTTCTCTTTC AG393_lnt_- 49_Fw_KpnI AAAAAggtaccACCCCAGCCGAAGCTGGATG AG403_lgt_myc CT_PstI AAAAACTGCAGctacaggtcttcttcgctaatcagtttctgttcgcttgcGGAAACGTGTT GCTGTGGGC PL387- LolBwoss-Fw- NcoI acacCCATGGccgttaccacgcccaaagg ColicinIalinker_ geneBLOCK acatccatggggATTCTGGACACGCGGTTGTCAGAGCTGGAAAAAAATG GCGGGGCAGCCCTTGCCGTTCTTGATGCACAACAGGCCCGTCTGC TCGGGCAGCAGACACGGAATGACAGGGCCATTTCAGAGGCACGG AATAAACTCAGTTCAGTGACGGAATCGCTTAACACGGCCCGTAAT 53 GCATTAACCAGAGCTGAACAACAGCTGACGCAACAGAAAgcggtccg acat 869 54