The Ubiquitin proteasome system (UPS) is responsible for tagging proteins with ubiquitin and targeting them for degradation. Multiple enzymes in this pathway carry out the sequential modification, processing, and degradation of proteins via UPS.[1] The degradation process is tightly regulated and ensures quality control.2, 3 Any dysregulation in the process can be deleterious leading to pathological conditions including neurodegenerative diseases [4], cell cycle dysregulation [5], inflammatory diseases 6, 7, cancer 8, 9 etc.

A sequential three-step cascade of enzymes facilitates the process of ubiquitylation. E3 ligases catalyse the final step of the ubiquitin conjugation process, i.e., covalent attachment of ubiquitin to the substrate.10, 11 This penultimate step is the most important step in terms of rendering substrate specificity, and there exists a large class of E3 ligases to efficiently catalyse this final step of Ubiquitin conjugation.[12] E3 ligases have been classified into three major families; namely Really Interesting New Gene (RING) family, Homology to E6AP C-terminus (HECT) and (Ring Between Ring) RBR family.13, 14

Among the RING E3 ligases, Skp1-Cullin1-F-box (SCF) RING E3 ligases are the largest RING E3 ligases, and their target substrates are actively involved in many key cellular processes like cell cycle regulation, signal transduction, and DNA replication.[15] Alterations in the ubiquitylation by the SCF ligases or their F-box proteins have resulted in genomic instability therefore these complexes are being actively pursued as anti-cancer targets.16, 17 SCF ligases consist of the following components: A central scaffold protein, Cullin; an adapter protein Skp1; Ring box protein (Rbx1); and substrate binding FBPs. The C-terminus of Cullin is the attachment site for Rbx1 which eventually binds to the E2-Ubiquitin-conjugating enzyme. The N-terminus of the Cullin is the binding site for Skp1 which binds with the substrate-binding proteins.18, 19, 20 Skp1 acts as an adapter in the SCF complex connecting the constant parts of the ligase to its variable substrate receptors, the F-box proteins. The human Skp1 is unique as it recognises 69 different F-box proteins and plays a critical role in ensuring fidelity in cell quality control.21, 22 Recent reports have suggested that reduced Skp1 expression is directly associated with the DNA double-strand break and chromosome instability leading to cancer 23, 24, 5, 25. A functional homologue of Skp1, Elongin C is another adapter protein that has been implicated in tumour suppressor activity and cell cycle regulation via VHL(Von Hippal-Lindau) complex.26, 27, 28

Previous structural studies have presented X-ray structures of the SCF complex, where Skp1 has been crystallized or complexed as part of the multiprotein E3-ligase complex.29, 30 These studies often used truncated or partially mutated protein constructs for structure determination by crystallography approaches.31, 32, 33 However, since the loops are structurally conserved across species, their functional aspects cannot be ignored.[34] To date, no standalone structure of Skp1 is available, which can be compared with the X-ray structures to determine the changes occurring in Skp1 upon F-box binding.

In this background, we report the structural and dynamic features of the human Skp1 investigated by solution-state NMR spectroscopy. Our results reveal a complex dynamic nature of the protein, displaying motions at different time scales in different portions of the chain. The dynamic nature of C-terminal helices could be a key feature of adapter proteins among the E3 ligase, which dictate the recognition and modulation of the conformational dynamics in order to bind F-box proteins and subsequent ubiquitylation. Comparison of the solution structure of the free protein with the structures of the protein in the different complexes reported earlier by different methods reveals residue-specific structural changes of C-terminal helices H6, H7, and H8 to account for adaptation upon substrate binding.

The NMR structure of Skp1 provides the three-dimensional structure of the native state of human origin Skp1. We have further explored the native state conformational ensemble by monitoring the amide proton chemical shift as a function of temperature. Small perturbations in temperature are often used to populate lowly-populated excited alternative conformational states in a protein. Since we noticed that Skp1 could alter its structure in different functional conformations from our structure, we explored the existence of alternative conformations in the native-state ensemble. These alternate states provide a glimpse into the functional states that protein might adopt during substrate binding.