| Summary: | The KDEL receptor (KDELR) is a trafficking receptor which is involved in the retrograde trafficking of ER resident proteins that escaped to the Golgi. For this function, the receptor cycles in COPI and COPII coated vesicles bet en Golgi, where cargo is bound, and the ER, where it is released. KDELR recognises its cargo by an ER retrieval sequence (ERS), the canonical signal consisting of a KDEL peptide motif. Cargo binding and dissociation is regulated by the pH difference between the two organelles. At low pH ERS binding is favoured whereas neutral pH triggers release. The crystal structures of the apo KDELR at pH 9.0 (‘inactive’ state) and the receptor bound to KDEL peptide at pH6.0 (‘active’ state) were solved recently. These revealed that the lysine-rich KDELR C-terminus forms the interface for COPI binding. A gain of structure transition of the C-terminus from ‘inactive’ to ‘active’ state is responsible for COPI recruitment to KDELR which initiates vesicle formation and retrograde trafficking. In this study, solution state NMR is used to investigate the molecular mechanism of KDELR2 signalling to COPI through its functionally important C-terminus. I aim to bridge the divide between the static crystal structures and the dynamic behaviour of the receptor in solution, which often holds the key to understand the function of a protein in the cell. Reductive methylation of lysine side chains with carbon-13 is used to introduce highly sensitive NMR active probes into the KDELR C-terminus. \textsuperscript{1}H-\textsuperscript{13}C correlation spectra were recorded of the receptor with the two variables of pH and presence of KDEL peptide. The NMR data revealed that pH by itself does not initiate KDELR signalling and solely regulates affinity of the receptor to peptide. Furthermore, I show that the KDELR C-terminus is highly dynamic in solution, sampling multiple conformations on the microsecond-millisecond timescale. In the absence of peptide, the C-terminus rapidly switches between its ‘active’ and ‘inactive’ states. Upon peptide binding the conformational equilibrium is shifted towards the ‘active’ state. However, a low level of conformational exchange remains. Based on these findings a model of KDELR signalling is proposed. The low pH in the Golgi primes the receptor for cargo binding, but does not initiate receptor signalling directly. The event of cargo binding is signalled across the membrane by the stabilisation of the ‘active’ conformation of the C-terminus. This leads to rapid recruitment of COPI and efficient retrograde trafficking to the ER. In case the Golgi is depleted of cargo, apo receptor is retained in the Golgi. The conformational flexibility and transient adoption of the 'active' conformation of the C-terminus in this state may allow for some signalling and a low level of receptor retrieval. This could prevent the receptor from following the bulk flow to the lysosome where it would get degraded. This study supports the hypothesis that protein dynamics is critical for KDELR function in the cell.
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