Phosphorylation at Ser65 modulates ubiquitin conformational dynamics

Highlights

• Transition between ubiquitin conformations involves a Bent intermediate

• Disrupting the hydrogen bond between pSer65 and Gln2 destabilizes the Bent state

• Decoupling between $\beta 5$ and $\beta 1$ strands accompanies the pUb Major to CR transition

Summary

Ubiquitin phosphorylation at Ser65 increases the population of a rare C-terminally retracted (CR) conformation. Transition between the Major and CR ubiquitin conformations is critical for promoting mitochondrial degradation. The mechanisms by which the Major and CR conformations of Ser65-phosphorylated (pSer65) ubiquitin interconvert, however, remain unresolved. Here, we perform all-atom molecular dynamics simulations using the string method with swarms of trajectories to calculate the lowest free-energy path between these two conformers. Our analysis reveals the existence of a Bent intermediate in which the C-terminal residues of the β5 strand shift to resemble the CR conformation, while pSer65 retains contacts resembling the Major conformation. This stable intermediate was reproduced in well-tempered metadynamics calculations but was less stable for a Gln2Ala mutant that disrupts contacts with pSer65. Lastly, dynamical network modeling reveals that the transition from the Major to CR conformations involves a decoupling of residues near pSer65 from the adjacent β1 strand.

Introduction

Ubiquitin (Ub) phosphorylation at Ser65 by protein kinase 1 (PINK1) initiates the mitophagy pathway through activation of the E3 ligase Parkin.^1,2,3,4,5,6 PINK1 aggregates on the cytosolic surface of depolarized mitochondria⁷ where it phosphorylates ubiquitin.¹ The phosphorylated ubiquitin (pUb) then binds to and activates Parkin,^4,5,6 which mediates the assembly of polyubiquitin chains on mitochondrial outer membrane proteins,⁸ recruiting autophagy receptors and ultimately forming the LC3-positive phagophore for mitochondrial degradation.^9,10,11 Dysfunction of PINK1 and Parkin are associated with early-onset autosomal-recessive Parkinson’s disease.^11,12,13 In addition, Ser65 pUb granules have been found in postmortem human brain samples that are increased by age and with Parkinson’s disease.^14,15

Although the first crystal structure of ubiquitin was published in 1985,¹⁶ recent NMR studies have revealed a new conformation of ubiquitin in which the C-terminal beta strand, $\beta$5, retracts by two amino acids.^17,18 Both ubiquitin and Ser65-phosphorylated (pSer65) ubiquitin exist in an equilibrium between this C-terminally retracted (CR) conformation and the Major (Maj) conformation captured by the original crystal structure. The two conformations primarily differ by the network of hydrogen bonds formed between strand $\beta$5 and adjacent strands, $\beta$1 and $\beta$3.¹⁷ Chemical exchange saturation transfer (CEST) NMR experiments report that in phosphorylated ubiquitin, the occupancies of the Major (pUb-Maj) and CR (pUb-CR) conformations are 70% and 30%, respectively, whereas in non-phosphorylated ubiquitin, the population of the CR conformation (Ub-CR) is only ∼0.68%.¹⁸ This indicates that phosphorylation at Ser65 modulates the conformational dynamics of ubiquitin. Transition between these two conformations is critical for initiating mitophagy; PINK1 phosphorylates ubiquitin at Ser65 in the CR conformation,¹⁸ while the Major conformation of pUb is required to bind to and activate Parkin.^6,18 Although previous work has identified a transition pathway between the Ub-Maj and Ub-CR conformations,¹⁹ little is known about the molecular mechanism driving the transition between the pUb-Maj and pUb-CR conformations. Understanding how pUb conformational dynamics differ from Ub at a molecular level would provide valuable insight into the unique role of pUb in pathogenesis.

Here, we compute the transition path between pUb-Maj and pUb-CR and identify important intermediates along the pathway. We then compute conformational free-energy landscapes for both pUb and Ub to probe how Ser65 phosphorylation affects the ubiquitin conformers that are sampled. Lastly, we explore how the extent of local dynamic coupling involving the residues of $\beta$5 plays an important role in driving the overall conformational transition.