The role of cryptic ancestral symmetry in histone folding mechanisms across Eukarya and Archaea.

Histones compact and store DNA in both Eukarya and Archaea, forming heterodimers in Eukarya and homodimers in Archaea. Despite this, the folding mechanism of histones across species remains unclear. Our study addresses this gap by investigating 11 types of histone and histone-like proteins across humans, Drosophila, and Archaea through multiscale molecular dynamics (MD) simulations, complemented by NMR and circular dichroism experiments. We confirm and elaborate on the widely applied "folding upon binding" mechanism of histone dimeric proteins and report a new alternative conformation, namely, the inverted non-native dimer, which may be a thermodynamically metastable configuration. Protein sequence analysis indicated that the inverted conformation arises from the hidden ancestral head-tail sequence symmetry underlying all histone proteins, which is congruent with the previously proposed histone evolution hypotheses. Finally, to explore the potential formations of homodimers in Eukarya, we utilized MD-based AWSEM and AI-based AlphaFold-Multimer models to predict their structures and conducted extensive all-atom MD simulations to examine their respective structural stabilities. Our results suggest that eukaryotic histones may also form stable homodimers, whereas their disordered tails bring significant structural asymmetry and tip the balance towards the formation of commonly observed heterotypic dimers.

Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation.

Post-translational modification of proteins with polyubiquitin chains is a critical cellular signaling mechanism in eukaryotes with implications in various cellular states and processes. Unregulated ubiquitin-mediated protein degradation can be detrimental to cellular homeostasis, causing numerous diseases including cancers. Recently, macrocyclic peptides were developed that selectively target long Lysine-48-linked polyubiquitin chains (tetra-ubiquitin) to inhibit ubiquitin-proteasome system, leading to attenuation of tumor growth in vivo. However, structural determinants of the chain length and linkage selectivity by these cyclic peptides remained unclear. Here, we uncover the mechanism underlying cyclic peptide's affinity and binding selectivity by combining X-ray crystallography, solution NMR, and biochemical studies. We found that the peptide engages three consecutive ubiquitins that form a ring around the peptide and determined requirements for preferential selection of a specific trimer moiety in longer polyubiquitin chains. The structural insights gained from this work will guide the development of next-generation cyclic peptides with enhanced anti-cancer activity.

Conformational Tuning Shapes the Balance between Functional Promiscuity and Specialization in Paralogous Plasmodium Acyl-CoA Binding Proteins.

Paralogous proteins confer enhanced fitness to organisms via complex sequence-conformation codes that shape functional divergence, specialization, or promiscuity. Here, we dissect the underlying mechanism of promiscuous binding versus partial subfunctionalization in paralogues by studying structurally identical acyl-CoA binding proteins (ACBPs) from Plasmodium falciparum that serve as promising drug targets due to their high expression during the protozoan proliferative phase. Combining spectroscopic measurements, solution NMR, SPR, and simulations on two of the paralogues, A16 and A749, we show that minor sequence differences shape nearly every local and global conformational feature. A749 displays a broader and heterogeneous native ensemble, weaker thermodynamic coupling and cooperativity, enhanced fluctuations, and a larger binding pocket volume compared to A16. Site-specific tryptophan probes signal a graded reduction in the sampling of substates in the holo form, which is particularly apparent in A749. The paralogues exhibit a spectrum of binding affinities to different acyl-CoAs with A749, the more promiscuous and hence the likely ancestor, binding 1000-fold stronger to lauroyl-CoA under physiological conditions. We thus demonstrate how minor sequence changes modulate the extent of long-range interactions and dynamics, effectively contributing to the molecular evolution of contrasting functional repertoires in paralogues.

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin.

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub2) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin.

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub 2 ) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub 2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub 2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub 2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

Reweighting methods for elucidation of conformation ensembles of proteins.

Proteins are inherently dynamic macromolecules that exist in equilibrium among multiple conformational states, and motions of protein backbone and side chains are fundamental to biological function. The ability to characterize the conformational landscape is particularly important for intrinsically disordered proteins, multidomain proteins, and weakly bound complexes, where single-structure representations are inadequate. As the focus of structural biology shifts from relatively rigid macromolecules toward larger and more complex systems and molecular assemblies, there is a need for structural approaches that can paint a more realistic picture of such conformationally heterogeneous systems. Here, we review reweighting methods for elucidation of structural ensembles based on experimental data, with the focus on applications to multidomain proteins.

Site-Specific Detection and Characterization of Ubiquitin Carbamylation.

The physiological consequences of varying in vivo CO2 levels point to a general mechanism for CO2 to influence cellular homeostasis beyond regulating pH. Aside from a few instances where CO2 has been observed to cause post-translational protein modification, by forming long-lived carbamates, little is known about how transitory and ubiquitous carbamylation events could induce a physiological response. Ubiquitin is a versatile protein involved in a multitude of cellular signaling pathways as polymeric chains of various lengths formed through one of the seven lysines or N-terminal amine. Unique polyubiquitin (polyUb) compositions present recognition signals for specific ubiquitin-receptors which enables this one protein to be involved in many different cellular processes. Advances in proteomic methods have allowed the capture and identification of protein carbamates in vivo, and Ub was found carbamylated at lysines K48 and K33. This was shown to negatively regulate ubiquitin-mediated signaling by inhibiting polyUb chain formation. Here, we expand upon these observations by characterizing the carbamylation susceptibility for all Ub amines simultaneously. Using NMR methods which directly probe 15N resonances, we determined carbamylation rates under various environmental conditions and related them to the intrinsic pKas. Our results show that the relatively low pKas for half of the Ub amines are correlated with enhanced susceptibility to carbamylation under physiological conditions. Two of these carbamylated amines, not observed by chemical capture, appear to be physiologically relevant post-translational modifications. These findings point to a mechanism for varying the levels of CO2 due to intracellular localization, cellular stresses, and metabolism to affect certain polyUb-mediated signaling pathways.

Tsg101/ESCRT-I recruitment regulated by the dual binding modes of K63-linked diubiquitin.

The ESCRT-I protein Tsg101 plays a critical role in viral budding and endocytic sorting. Although Tsg101 is known to recognize monoubiquitin (Ub1), here we show that it can also bind several diubiquitins (K48-Ub2, N-Ub2, and K63-Ub2), with a preference for K63-linked Ub2. The NMR structure of the Tsg101:K63-Ub2 complex showed that while the Ub1-binding site accommodates the distal domain of Ub2, the proximal domain alternatively binds two different sites, the vestigial active site and an N-terminal helix. Mutation of each site results in distinct phenotypes regarding the recruitment of Tsg101 partners. Mutation in the vestigial active site abrogates interaction between Tsg101 and the HIV-1 protein Gag but not Hrs, a cellular protein. Mutation at the N-terminal helix alters Gag but not Hrs-Tsg101 localization. Given the broad involvement of Tsg101 in diverse cellular functions, this discovery advances our understanding of how the ESCRT protein recognizes binding partners and sorts endocytic cargo.

Backbone NMR resonance assignment of the intrinsically disordered UBact protein from Nitrospira nitrosa.

Ubiquitin signaling in eukaryotes is responsible for a variety of cellular outcomes, most notably proteasomal degradation. A recent bioinformatic study has revealed the existence of a new proteasomal operon in certain gram-negative bacteria phyla. This operon contains genes similar to those included in the prokaryotic ubiquitin-like protein (Pup) proteasomal operon, but do not themselves contain Pup. Instead, they encode for a protein termed UBact with 30% sequence similarity to Pup. Here, we report the near-complete NMR assignment of the backbone and partial assignment of the side chain chemical shifts of the UBact protein from Nitrospira nitrosa. The 1H-15N HSQC spectrum shows a narrow spread of proton NMR signals, characteristic of an intrinsically disordered protein. This chemical shift assignment will facilitate further NMR studies to explore the role of UBact in this new putative proteasomal operon.

Quantum computation of molecular structure using data from challenging-to-classically-simulate nuclear magnetic resonance experiments.

We propose a quantum algorithm for inferring the molecular nuclear spin Hamiltonian from time-resolved measurements of spin-spin correlators, which can be obtained via nuclear magnetic resonance (NMR). We focus on learning the anisotropic dipolar term of the Hamiltonian, which generates dynamics that are challenging to classically simulate in some contexts. We demonstrate the ability to directly estimate the Jacobian and Hessian of the corresponding learning problem on a quantum computer, allowing us to learn the Hamiltonian parameters. We develop algorithms for performing this computation on both noisy near-term and future fault-tolerant quantum computers. We argue that the former is promising as an early beyond-classical quantum application since it only requires evolution of a local spin Hamiltonian. We investigate the example of a protein (ubiquitin) confined on a membrane as a benchmark of our method. We isolate small spin clusters, demonstrate the convergence of our learning algorithm on one such example, and then investigate the learnability of these clusters as we cross the ergodic to non-ergodic phase transition by suppressing the dipolar interaction. We see a clear correspondence between a drop in the multifractal dimension measured across many-body eigenstates of these clusters, and a transition in the structure of the Hessian of the learning cost function (from degenerate to learnable). Our hope is that such quantum computations might enable the interpretation and development of new NMR techniques for analyzing molecular structure.

Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions.

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently, it was reported that UCH37 activity is stimulated by branched ubiquitin (Ub) chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and Nuclear Magnetic Resonance (NMR) structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal Ubs that emanate from a branched Ub. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear Ub chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome for the next round of substrate processing. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique Ub chain architecture is aided by a DUB.

Ubiquitin is a carbon dioxide-binding protein.

The identification of CO2-binding proteins is crucial to understanding CO2-regulated molecular processes. CO2 can form a reversible posttranslational modification through carbamylation of neutral N-terminal α-amino or lysine ε-amino groups. We have previously developed triethyloxonium (TEO) ion as a chemical proteomics tool for covalent trapping of carbamates, and here, we deploy TEO to identify ubiquitin as a mammalian CO2-binding protein. We use 13C-NMR spectroscopy to demonstrate that CO2 forms carbamates on the ubiquitin N terminus and ε-amino groups of lysines 6, 33, 48, and 63. We demonstrate that biologically relevant pCO2 levels reduce ubiquitin conjugation at lysine-48 and down-regulate ubiquitin-dependent NF-κB pathway activation. Our results show that ubiquitin is a CO2-binding protein and demonstrates carbamylation as a viable mechanism by which mammalian cells can respond to fluctuating pCO2.

A novel recognition site for polyubiquitin and ubiquitin-like signals in an unexpected region of proteasomal subunit Rpn1.

The ubiquitin (Ub)-proteasome system is the primary mechanism for maintaining protein homeostasis in eukaryotes, yet the underlying signaling events and specificities of its components are poorly understood. Proteins destined for degradation are tagged with covalently linked polymeric Ub chains and subsequently delivered to the proteasome, often with the assistance of shuttle proteins that contain Ub-like domains. This degradation pathway is riddled with apparent redundancy-in the form of numerous polyubiquitin chains of various lengths and distinct architectures, multiple shuttle proteins, and at least three proteasomal receptors. Moreover, the largest proteasomal receptor, Rpn1, contains one known binding site for polyubiquitin and shuttle proteins, although several studies have recently proposed the existence of an additional uncharacterized site. Here, using a combination of NMR spectroscopy, photocrosslinking, mass spectrometry, and mutagenesis, we show that Rpn1 does indeed contain another recognition site that exhibits affinities and binding preferences for polyubiquitin and Ub-like signals comparable to those of the known binding site in Rpn1. Surprisingly, this novel site is situated in the N-terminal section of Rpn1, a region previously surmised to be devoid of functionality. We identified a stretch of adjacent helices as the location of this previously uncharacterized binding site, whose spatial proximity and similar properties to the known binding site in Rpn1 suggest the possibility of multivalent signal recognition across the solvent-exposed surface of Rpn1. These findings offer new mechanistic insights into signal recognition processes that are at the core of the Ub-proteasome system.

Correction: In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides.

[This corrects the article DOI: 10.1039/D0CB00179A.].

In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides.

Cancer and other disease states can change the landscape of proteins post-translationally tagged with ubiquitin (Ub) chains. Molecules capable of modulating Ub chains are potential therapeutic agents, but their discovery represents a significant challenge. Recently, it was shown that de novo cyclic peptides, selected from trillion-member random libraries, are capable of binding particular Ub chains. However, these peptides were overwhelmingly proteinogenic, so the prospect of in vivo activity was uncertain. Here, we report the discovery of small, non-proteinogenic cyclic peptides, rich in non-canonical features like N-methylation, which can tightly and specifically bind Lys48-linked Ub chains. These peptides engage three Lys48-linked Ub units simultaneously, block the action of deubiquitinases and the proteasome, induce apoptosis in vitro, and attenuate tumor growth in vivo. This highlights the potential of non-proteinogenic cyclic peptide screening to rapidly find in vivo-active leads, and the targeting of ubiquitin chains as a promising anti-cancer mechanism of action.

15N NMR studies provide insights into physico-chemical properties of room-temperature ionic liquids.

Ionic liquids (ILs) have gained a lot of attention as alternative solvents in many fields of science in the last two decades. It is known that the type of anion has a significant influence on the macroscopic properties of the IL. To gain insights into the molecular mechanisms responsible for these effects it is important to characterize these systems at the microscopic level. Such information can be obtained from nuclear spin-relaxation studies which for compounds with natural isotope abundance are typically performed using direct 1H or 13C measurements. Here we used direct 15N measurements to characterize spin relaxation of non-protonated nitrogens in imidazolium-based ILs which are liquid at ambient temperature. We report heteronuclear 1H-15N scalar coupling constants (nJHN) and 15N relaxation parameters for non-protonated nitrogens in ten 1-ethyl-3-methylimidazolium ([C2C1IM]+)-based ILs containing a broad range of anions. The 15N relaxation rates and steady-state heteronuclear 15N-{1H} NOEs were measured using direct 15N detection at 293.2 K and two magnetic field strengths, 9.4 T and 16.4 T. The experimental data were analyzed to determine hydrodynamic characteristics of ILs and to assess the contributions to 15N relaxation from 15N chemical shift anisotropy and from 1H-15N dipolar interactions with non-bonded protons. We found that the rotational correlation times of the [C2C1IM]+ cation determined from 15N relaxation measurements at room temperature correlate linearly with the macroscopic viscosity of the ILs. Depending on the selected anion, the 15N relaxation characteristics of [C2C1IM]+ differ considerably reflecting the influence of the anion on the physicochemical properties of the IL.

Polyubiquitin and ubiquitin-like signals share common recognition sites on proteasomal subunit Rpn1.

Proteasome-mediated substrate degradation is an essential process that relies on the coordinated actions of ubiquitin (Ub), shuttle proteins containing Ub-like (UBL) domains, and the proteasome. Proteinaceous substrates are tagged with polyUb and shuttle proteins, and these signals are then recognized by the proteasome, which subsequently degrades the substrate. To date, three proteasomal receptors have been identified, as well as multiple shuttle proteins and numerous types of polyUb chains that signal for degradation. While the components of this pathway are well-known, our understanding of their interplay is unclear-especially in the context of Rpn1, the largest proteasomal subunit. Here, using nuclear magnetic resonance (NMR) spectroscopy in combination with competition assays, we show that Rpn1 associates with UBL-containing proteins and polyUb chains, while exhibiting a preference for shuttle protein Rad23. Rpn1 appears to contain multiple Ub/UBL-binding sites, theoretically as many as one for each of its hallmark proteasome/cyclosome repeats. Remarkably, we also find that binding sites on Rpn1 can be shared among Ub and UBL species, while proteasomal receptors Rpn1 and Rpn10 can compete with each other for binding of shuttle protein Dsk2. Taken together, our results rule out the possibility of exclusive recognition sites on Rpn1 for individual Ub/UBL signals and further emphasize the complexity of the redundancy-laden proteasomal degradation pathway.

SOS1 interacts with Grb2 through regions that induce closed nSH3 conformations.

Grb2 is an adaptor protein connecting the epidermal growth factor receptor and the downstream Son of sevenless 1 (SOS1), a Ras-specific guanine nucleotide exchange factor (RasGEF), which exchanges GDP by GTP. Grb2 contains three SH domains: N-terminal SH3 (nSH3), SH2, and C-terminal SH3 (cSH3). The C-terminal proline-rich (PR) domain of SOS1 regulates nSH3 open/closed conformations. Earlier, several nSH3 binding motifs were identified in the PR domain. More recently, we characterized by nuclear magnetic resonance and replica exchange simulations possible cSH3 binding regions. Among them, we discovered a cSH3-specific binding region. However, how PR binding at these sites regulates the nSH3/cSH3 conformation has been unclear. Here, we explore the nSH3/cSH3 interaction with linked and truncated PR segments using molecular dynamics simulations. Our 248 µs simulations include 620 distinct trajectories, each 400 ns. We construct the effective free energy landscape to validate the nSH3/cSH3 binding sites. The nSH3/cSH3-SOS1 peptide complex models indicate that strong peptide binders attract the flexible nSH3 n-Src loop, inducing a closed conformation of nSH3; by contrast, the cSH3 conformation remains unchanged. Inhibitors that disrupt the Ras-SOS1 interaction have been designed; the conformational details uncovered here may assist in the design of polypeptides inhibiting Grb2-SOS1 interaction, thus SOS1 recruitment to the membrane where Ras resides.

Mechanism of Catalysis by l-Asparaginase.

Two bacterial type II l-asparaginases, from Escherichia coli and Dickeya chrysanthemi, have played a critical role for more than 40 years as therapeutic agents against juvenile leukemias and lymphomas. Despite a long history of successful pharmacological applications and the apparent simplicity of the catalytic reaction, controversies still exist regarding major steps of the mechanism. In this report, we provide a detailed description of the reaction catalyzed by E. coli type II l-asparaginase (EcAII). Our model was developed on the basis of new structural and biochemical experiments combined with previously published data. The proposed mechanism is supported by quantum chemistry calculations based on density functional theory. We provide strong evidence that EcAII catalyzes the reaction according to the double-displacement (ping-pong) mechanism, with formation of a covalent intermediate. Several steps of catalysis by EcAII are unique when compared to reactions catalyzed by other known hydrolytic enzymes. Here, the reaction is initiated by a weak nucleophile, threonine, without direct assistance of a general base, although a distant general base is identified. Furthermore, tetrahedral intermediates formed during the catalytic process are stabilized by a never previously described motif. Although the scheme of the catalytic mechanism was developed only on the basis of data obtained from EcAII and its variants, this novel mechanism of enzymatic hydrolysis could potentially apply to most (and possibly all) l-asparaginases.