Leo Spyracopoulos's research while affiliated with University of Alberta and other places

Protein turnover in cells is regulated by the ATP dependent activity of the Hsp90 chaperone. In concert with accessory proteins, ATP hydrolysis drives the obligate Hsp90 dimer through a cycle between open and closed states that is critical for assisting the folding and stability of hundreds of proteins. Cycling is initiated by ATP binding to the ATPase domain, with the chaperone and the active site gates in the dimer in open states. The chaperone then adopts a short-lived, ATP bound closed state with a closed active site gate. The structural and dynamic changes induced in the ATPase domain and active site gate upon nucleotide binding, and their impact on dimer closing are not well understood. We site-specifically ¹⁹F-labeled the ATPase domain at the active site gate to enable benchtop and high field ¹⁹F NMR spectroscopic studies. Combined with MD simulations, this allowed accurate characterization of pico- to nanosecond timescale motions of the active site gate, as well as slower micro- to millisecond timescale processes resulting from nucleotide binding. ATP binding induces increased flexibility at one of the hinges of the active site gate, a necessary prelude to release of the second hinge, and eventual gate closure in the intact chaperone.

F NMR spectroscopy is a powerful tool for the study of the structures, dynamics, and interactions of proteins bearing cysteine residues chemically modified with a trifluoroacetone group (CYF residue). ¹⁹ F NMR relaxation rates for the fluoromethyl group of CYF residues are sensitive to overall rotational tumbling of proteins, fast rotation about the CF 3 methyl axis, and the internal motion of the CYF side-chain. To develop a quantitative understanding of these various motional contributions, we used the model-free approach to extend expressions for ¹⁹ F-T 2 NMR relaxation to include side-chain motions for the CYF residue. We complemented the NMR studies with atomic views of methyl rotation and side-chain motions using molecular dynamics simulations. This combined methodology allows for quantitative separation of the contributions of fast pico- to nanosecond dynamics from micro- to millisecond exchange processes to the ¹⁹ F line width and highlights the utility of the CYF residue as a sensitive reporter of side-chain environment and dynamics in proteins.

Hsp90 is a crucial chaperone whose ATPase activity is fundamental for stabilizing and activating a diverse array of client proteins. Binding and hydrolysis of ATP by dimeric Hsp90 drives a conformational cycle characterized by fluctuations between a compact, N- and C-terminally dimerized catalytically competent closed state, and less compact open state which is largely C-terminally dimerized. We used ¹⁹F and ¹H dynamic NMR spectroscopy to study the opening and closing kinetics of Hsp90, and to determine the kcat for ATP hydrolysis. We derived a set of coupled ordinary differential equations describing the rate laws for the Hsp90 kinetic cycle, and used these to analyze the NMR data. We find that the kinetics of closing and opening for the chaperone are slow, and that the lower limit for kcat of ATP hydrolysis is ~1 s–1. Our results show that the chemical step is optimized, and that Hsp90 is indeed, a “perfect” enzyme.

Cullin-RING ubiquitin ligases are a diverse family of ubiquitin ligases that catalyze the synthesis of K48-linked polyubiquitin (polyUb) chains on a variety of substrates, ultimately leading to their degradation by the proteasome. The cullin-RING enzyme scaffold processively attaches a Ub molecule to the distal end of a growing chain up to lengths of eight Ub monomers. However, the molecular mechanism governing how chains of increasing size are built using a scaffold of largely fixed dimensions is not clear. We developed coarse-grained (CG) molecular dynamics simulations to describe the dependence of kcat for cullin-RING ligases on the length and flexibility of the K48-linked polyUb chain attached to the substrate protein, key factors that determine the rate of subsequent Ub attachment to the chain, and therefore, the ensuing biological outcomes of ubiquitination. The results suggest that a number of regulatory mechanisms may lead to variations in the rate of chain elongation for different cullin-RING ligases. Specifically, modulation of the distance between the target lysine and the phosphodegron sequence of the substrate, the distance between the substrate lysine and the active site cysteine of the Ub conjugation enzyme (E2) bound to the cullin-RING scaffold, and flexibility of the bound E2, can lead to significant differences in the processing of K48-linked chains on substrates, potentially leading to differences in biological outcomes.

Interleukin-8 (CXCL8), a potent neutrophil-activating chemokine, exerts its function by activating the CXCR1 receptor that belongs to class A G protein-coupled receptors (GPCRs). Receptor activation involves interactions between the CXCL8 N-terminal loop and CXCR1 N-terminal domain (N-domain) residues (Site-I) and between the CXCL8 N-terminal and CXCR1 extracellular/transmembrane residues (Site-II). CXCL8 exists in equilibrium between monomers and dimers, and it is known that the monomer binds CXCR1 with much higher affinity and that Site-I interactions are largely responsible for the differences in monomer vs. dimer affinity. Here, using backbone 15N-relaxation nuclear magnetic resonance (NMR) data, we characterized the dynamic properties of the CXCL8 monomer and the CXCR1 N-domain in the free and bound states. The main chain of CXCL8 appears largely rigid on the picosecond time scale as evident from high order parameters (S2). However, on average, S2 are higher in the bound state. Interestingly, several residues show millisecond-microsecond (ms-μs) dynamics only in the bound state. The CXCR1 N-domain is unstructured in the free state but structured with significant dynamics in the bound state. Isothermal titration calorimetry (ITC) data indicate that both enthalpic and entropic factors contribute to affinity, suggesting that increased slow dynamics in the bound state contribute to affinity. In sum, our data indicate a critical and complex role for dynamics in driving CXCL8 monomer-CXCR1 Site-I interactions.

The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.

Ring1-YY1-binding protein (RYBP) is a member of the non-canonical polycomb repressive complex 1 (PRC1), and like other PRC1 members, it is best described as a transcriptional regulator. However, several PRC1 members were recently shown to function in DNA repair. Here, we report that RYBP preferentially binds K63-ubiquitin chains via its Npl4 zinc finger (NZF) domain. Since K63-linked ubiquitin chains are assembled at DNA double-strand breaks (DSBs), we examined the contribution of RYBP to DSB repair. Surprisingly, we find that RYBP is K48 polyubiquitylated by RNF8 and rapidly removed from chromatin upon DNA damage by the VCP/p97 segregase. High expression of RYBP competitively inhibits recruitment of BRCA1 repair complex to DSBs, reducing DNA end resection and homologous recombination (HR) repair. Moreover, breast cancer cell lines expressing high endogenous RYBP levels show increased sensitivity to DNA-damaging agents and poly ADP-ribose polymerase (PARP) inhibition. These data suggest that RYBP negatively regulates HR repair by competing for K63-ubiquitin chain binding.

Cells are exposed to thousands of DNA damage events on a daily basis. This damage must be repaired to preserve genetic information, and prevent development of disease. The most deleterious damage is a double strand break (DSB), which is detected and repaired by mechanisms known as non-homologous end joining (NHEJ), and homologous recombination (HR), components of the DNA damage response system. NHEJ is an error prone first line of defense, whereas HR invokes error free repair, and is the focus of this review. The functions of the protein components of HR-driven DNA repair are regulated by the coordinated action of post-translational modifications including lysine acetylation, phosphorylation, ubiquitination, and SUMOylation. The latter two mechanisms are fundamental for recognition of DSBs, and reorganizing chromatin to facilitate repair. We focus on the structures and molecular mechanisms for the protein components underlying synthesis, recognition, and cleavage of K63-linked ubiquitin chains, which are abundant at damage sites, and obligatory for DSB repair. The forward flux of the K63-linked ubiquitination cascade is driven by the combined activity of E1 enzyme, the heterodimeric E2 Mms2-Ubc13, and its cognate E3 ligases RNF8/RNF168, which is balanced through the binding and cleavage of chains by the deubiquitinase BRCC36 and the proteasome, as well as binding of chains by recognition modules on repair proteins such as RAP80. We highlight a number of aspects regarding our current understanding for the role of kinetics and dynamics in determining the function of the enzymes and chain recognition modules that drive K63-ubiquitination.

Hsp90 is a dimeric molecular chaperone responsible for the folding, maturation, and activation of hundreds of substrate proteins called ‘clients’. Numerous co-chaperone proteins regulate progression through the ATP-dependent client activation cycle. The most potent stimulator of the Hsp90 ATPase activity is the co-chaperone Aha1p. Only one molecule of Aha1p is required to fully stimulate the Hsp90 dimer despite the existence of two, presumably identical, binding sites for this regulator. Using ATPase assays with Hsp90 heterodimers, we find that Aha1p stimulates ATPase activity by a three-step mechanism via the catalytic loop in the middle domain of Hsp90. Binding of the Aha1p N domain to the Hsp90 middle domain exerts a small stimulatory effect but also drives a separate conformational rearrangement in the Hsp90 N domains. This second event drives a rearrangement in the N domain of the opposite subunit and is required for the stimulatory action of the Aha1p C domain. Furthermore, the second event can be blocked by a mutation in one subunit of the Hsp90 dimer but not the other. This work provides a foundation for understanding how post-translational modifications regulate co-chaperone engagement with the Hsp90 dimer.