A baton-relay and proofreading mechanism for selective ER retrieval signal capture by the KDEL receptor

The KDEL-retrieval pathway captures escaped ER proteins with a KDEL or variant C-terminal signal at acidic pH in the Golgi and releases them at neutral pH in the ER. To address the mechanism of signal binding and the molecular basis for differences in signal affinity, we determined the HDEL and RDEL bound structures of the KDEL-receptor. Affinity differences are explained by interactions between the variable −4 position of the signal and W120, whereas initial capture of retrieval signals by their carboxyl-terminus is mediated by a baton-relay mechanism involving a series of conserved arginine residues in the receptor. This explains how the signal is first captured and then pulled into the binding cavity. During capture, retrieval signals undergo a selective proofreading step involving two gatekeeper residues D50 and E117 in the receptor. These mechanisms operate upstream of the pH-dependent closure of the receptor and explain the selectivity of the KDEL-retrieval pathway.

carboxyl-terminus is mediated by a baton-relay mechanism involving a series of 23 conserved arginine residues in the receptor. This explains how the signal is first 24 captured and then pulled into the binding cavity. During capture, retrieval signals 25 undergo a selective proofreading step involving two gatekeeper residues D50 and 26 E117 in the receptor. These mechanisms operate upstream of the pH-dependent 27 closure of the receptor and explain the selectivity of the KDEL-retrieval pathway. 28 INTRODUCTION 30 Stable maintenance of the luminal composition of the endoplasmic reticulum (ER) is 31 crucial for the function of the secretory pathway (Ellgaard and Helenius, 2003). 32 COS7 cells (Lewis and Pelham, 1992;Wilson et al., 1993). However, we find that 117 DDEL binds with 60-fold lower affinity than HDEL (K D 14.9 µM) (Figure 1c), in 118 agreement with other data for purified KDEL receptors (Scheel and Pelham, 1998). 119 Thus, the receptor binds to the HDEL sequence with one order of magnitude greater Given its higher affinity, why then is HDEL not the dominant ER retrieval 132 signal, especially for crucial ER proteins such as BIP, PDI and calreticulin? We 133 tested the idea that due to its higher binding affinity, increasing the concentration of 134 HDEL bearing proteins would effectively compete for KDEL receptors in the Golgi, 135 and prevent efficient ER retrieval of KDEL and RDEL containing proteins. To do this 136 we used our series of variant xDEL signals, where x at the -4 position is either K, R, 137 H, A or D. When expressed in cells, KDEL, RDEL and HDEL are retained in the cell, 138 whereas ADEL and DDEL are mostly secreted (Figure 1supplement 1d and e). 139 With the exception of HDEL this is broadly in line with their respective binding 140 affinities. Despite binding to the receptor with a higher affinity (Figure 1c), HDEL was 141 less efficiently retained than either KDEL or RDEL (Figure 1 supplement 1d and e). 142 We then examined the effect of these ligands on the major ER proteins BIP and PDI 143 as well as the less abundant chaperones ERP72 and ERP44 ((Figure 1 -144 supplement 1a). As predicted, ADEL and DDEL had little effect on ER retention, 145 while HDEL caused secretion of all four proteins (Figure 1supplement 1f). 146 These results indicate that the retrieval system is selective yet not optimised 147 for binding affinity, and instead has evolved to ensure optimal retrieval of a broad 148 cohort of proteins of widely differing abundance. In human cells, ADEL and DDEL do 149 not bind to the receptor with high affinity and do not function as retrieval signals, 150 suggesting specific recognition of the -4 position is a key determinant for binding. 151 Previously, it has been suggested that complementary charges at receptor position 152 50 and the -4 position of the signal explain this specificity (Lewis and Pelham, 1992; 153 Semenza and Pelham, 1992). However, this mechanism does not obviously explain 154 how ADEL, with no charged residue at the -4 position, functions as a signal in some 155 organisms. How signal selectivity is achieved is therefore a crucial question we need 156 to answer. 159 To understand the molecular basis for the affinity differences between retrieval signal  Table S1). In both 163 instances the overall structure of the receptor is similar to our previous complex with 1a). In our structures, the glutamate at the -5 position sits close to S54, but would not 184 obviously increase the binding affinity, whereas no contacts are made to the -6 185 position. In all cases, the -1 position leucine residue and free carboxy terminus form 186 interactions to R47 and Y48 on TM2, as well as R159 and Y162 on TM6. The 187 glutamate at position -2 forms a further salt bridge interaction to R5 on TM1 and a 188 hydrogen bond to W166 on TM6, whereas the aspartate at -3 forms a salt bridge 189 with R169, also on TM6. For the histidine side chain at the -4 position of HDEL, the 190 imidazole group is predicted to form a - stacking interaction with W120 ( Figure  2b). In comparison, the RDEL arginine side chain sits in the same position as the 192 amine group of the KDEL sequence and could thus interact with W120 via a cation- 193 interaction and E117 via a classical salt bridge interaction (Figure 2c). We therefore 194 conclude that both E117 and W120 play a role in retrieval signal binding, and the 195 only major difference between the HDEL, RDEL and KDEL signals is the precise 196 nature of the interaction with W120 indicating that this may be a critical residue to 197 explain the affinity of different signals. 198 199 Probing the importance of E117 and W120 for signal binding 200 To directly test the requirement for E117 and W120 in signal recognition, ligand 201 binding assays using recombinant chicken wild type, E117 or W120 mutant KDELR2  (Figure 3a and 3b). This suggested that any salt bridge to E117 plays a 210 greater role for KDEL than HDEL signals. 211 We next examined the contribution of W120 to signal recognition. Tryptophan 212 side chains have long been recognized as important contributors in protein ligand 213 interactions, as they are capable of interacting with ligands via both aromatic and 214 charged forces (Dougherty, 1996;Liao et al., 2013;Okada et al., 2001). Our 215 structures show that the -4 position histidine, arginine or lysine side chain of the 216 human retrieval signal variants can in principle interact favourably with W120 via 217 cation- interactions. We also reasoned that given the additional - stacking 218 observed with the imidazole group in the crystal structure, this interaction might 219 explain the increased affinity observed for the HDEL signal variant. Mutation of 220 W120 to alanine resulted in loss of binding to the KDEL peptide and it was not 221 possible to calculate a K D (Figure 3a). For the HDEL peptide, binding was reduced to 222 20% confirming that W120 plays an important role in mediating receptor-peptide 223 interactions ( Figure 3b). Consistent with the hypothesis that the -4 histidine of HDEL 224 undergoes - stacking interactions with W120, conserved substitution to 225 phenylalanine supported 50% HDEL binding with K D 5.5 ± 0.57 µM, whereas no 226 interaction was observed with the KDEL peptide (Figure 3a and 3b). Thus, W120 227 plays a crucial role in binding of both KDEL and HDEL signals and may explain the 228 higher affinity of the receptor for HDEL. By contrast, E117 is less important and it is 229 unclear why it is a conserved feature of the binding site. 230 To analyse whether the properties measured using purified components in 231 vitro reflect the behaviour of the KDEL receptor and retrieval system in vivo, we 232 analysed the ability of these same variants in the human KDEL receptor to 233 differentiate between human retrieval signal sequences in a cellular ER retrieval    (Table S2), which is in good agreement with the expected -1.3 257 kcal/mol free energy difference derived from measured K D values for KDEL and 258 HDEL. The preference for HDEL of -1.9  0.2 kcal.mol -1 is mainly attributed to the 259 protonated histidine which makes favourable cation- interactions with W120 (Table   260 S2, HIP). In agreement with the experimental data ( Figure 3b and 3c), the W120F 261 mutation, which is anticipated to preserve the cation- interactions, reduced but did 262 not abolish the preference for HDEL to -0.7  1.6 kcal.mol -1 , notwithstanding the 263 large error on this calculation. Furthermore, the W120A mutation which eliminates 264 the cation- interactions, greatly reduced the preference for HDEL to -0.3  0.9 265 kcal.mol -1 .

HDEL and RDEL signals bind similarly to the canonical KDEL variant
To quantify the strength of the - and cation- interactions between W120 267 variants and the histidine, we decomposed the interactions using symmetry-adapted 268 perturbation theory from quantum mechanics. Although both W120 and W120F form 269 - and cation- interactions with protonated histidine, W120F exhibits ~1.5 kcal/mol 270 weaker - interactions and ~0.5 kcal/mol weaker cation- interactions with the 271 histidine ( Figure 3d and Table S3). The consequence of these changes is that for This mode of signal binding involving W120 is different than previously proposed, 288 where charge complementarity between D50 in the receptor and the -4 position of 289 the signal was thought to be a key determinant of specificity in ER retrieval (Lewis 290 and Pelham, 1992; Scheel and Pelham, 1998; Semenza and Pelham, 1992).
However, as our crystal structures show, D50 is outside the immediate binding 292 region for all retrieval signal variants and therefore unlikely to directly contribute to 293 binding. Thus, the precise roles of D50 and E117 remain enigmatic. In this regard 294 the behaviour of ADEL signals is noteworthy due to the simple methyl side chain. 295 Comparison of different retrieval signals shows that ADEL does not activate the wild 296 type human KDEL receptor (Figure 1d and 1e). The simplest explanation for this 297 finding is that the -4 position is crucial for high affinity binding of retrieval signals to 298 the human receptor. However, this view is unlikely to be correct. First, the KDEL, 299 RDEL and HDEL bound receptor structures do not support the view that recognition 300 of the -4 position requires D50, and instead provide an alternative possibility where 301 E117 fulfils this role. However, our biochemical and functional data show that E117 302 does not contribute greatly to signal binding affinity or retrieval in cells (Figure 3a-303 3c). Therefore, rather than selecting for the sequence, E117 may be more important 304 to select against unwanted signal variants. To test this idea, we examined the 305 response of E117A mutant receptors to variant ADEL and DDEL signals. 306 Remarkably, the E117A mutant receptor relocated to the ER in response to both 307 KDEL and ADEL, but not DDEL signals (Figure 4a and 4b). In S. pombe and K. 308 lactis, organisms where ADEL and DDEL are used for ER retrieval, the E117 309 position is either an asparagine or a glutamine residue, and we therefore tested Thus, E117 is important for determining which signals are rejected by the wild 317 type human receptor based on the -4 position of the signal, but does not appear to 318 play a major role in binding affinity. ADEL must bind to the E117A mutant receptors 319 via the "DEL" tri-carboxylate portion of the retrieval signal, suggesting this region 320 may be the major contributor to binding affinity for all signal variants. For HDEL, the 321 protonated histidine side chain makes additional - interactions with W120 to bind 322 with higher affinity. Importantly, the lack of response to DDEL shows that signal 323 selection and recognition must involve additional features in the S. pombe and K. 324 lactis receptor, and we investigated this question further.  383 These results indicate that the -4 position of the signal is read out during initial 384 signal binding and is important for exclusion of unwanted signals, but is less 385 important for binding affinity. We therefore tested whether the mode of ADEL and 386 DDEL binding to the switched specificity receptors still involves W120. A D50N N54K 387 E117Q W120A mutant receptor does not relocate from the Golgi to the ER with 388 KDEL and ADEL signals and shows only a small response to the DDEL signal 389 (Figure 6a and 6b). Together, these findings suggest a common mode of binding for 390 all retrieval signal variants through residues conserved in all species. Specificity for the -4 position is largely achieved through a proofreading mechanism involving two 392 gatekeeper residues, D50 and E117, as the signal enters the ligand binding cavity. 393 An E117A substitution partially uncouples this mechanism and allows ADEL binding, 394 whereas both D50 and E117 residues have to be changed to allow DDEL binding. 395 Bringing together all our observations to this point, we conclude that the luminal

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A baton-relay mechanism for initial signal capture by the KDEL receptor 446 Canonical ER retrieval signals can be broken down into two components: the -4 447 position, which enables the receptor to distinguish between different populations of 448 ER proteins, and a tri-carboxylate moiety formed by the -3 aspartate, -2 glutamate 449 and -1 C-terminal carboxylate. We propose a baton-relay handover mechanism for 450 capture of this signal by the KDEL receptor wherein a ladder of three arginine 451 residues in the receptor pairs with the three-carboxyl groups of the signal (Figure 8). 452 During cargo capture, the receptor engages the retrieval signal in a stepwise 453 process, with the C-terminal carboxyl group of the cargo protein moving between 454 these three interaction sites. At neutral pH, C-terminal sequences will rapidly sample 455 the binding site, a process that we imagine will occur in both the ER and Golgi where the signal and receptor are dimerised by a split YFP molecule that will likely 486 contribute to the observed signal binding affinity. This will interfere with the initial 487 proofreading mechanism described here, making comparison with our data difficult. 488 Since the three human KDEL receptors share identical ligand binding residues and 489 only differ in conservative substitutions at position 54, we believe they will have the 490 same or closely-related ligand binding properties. Based on the structures it seems 491 reasonable that the -5 position may contribute to signal proofreading in some cases.
However, as we show, a wide variety of signals that lack any obvious conserved 493 features upstream of the canonical tetrapeptide function efficiently to trigger ER 494 retrieval of the receptor (Figure 1a and S5b-S5e), suggesting the -5 position 495 modulates but does not play an essential role in signal recognition. Taken together, 496 these data support a model for retrieval sequence recognition that explains both the 497 importance of the free C-terminal carboxyl group and how changes at the -4 position 498 can modulate binding to the receptor.