Inherited mutations in human beta-cardiac myosin (M2β) can lead to severe forms of heart failure. The E525K mutation in M2β is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2β subfragment 1 (S1) we found that E525K enhances (threefold) the maximum steady-state actin-activated ATPase activity (k cat) and decreases (eightfold) the actin concentration at which ATPase is one-half maximal (K ATPase). We also found a twofold to fourfold increase in the actin-activated power stroke and phosphate release rate constants at 30 μM actin, which overall enhanced the duty ratio threefold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2β S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis.

Human somatic angiotensin-converting enzyme (ACE) is a key zinc metallopeptidase that plays a pivotal role in the renin-angiotensin-aldosterone system (RAAS) by regulating blood pressure and electrolyte balance. Inhibition of ACE is a cornerstone in the management of hypertension, cardiovascular diseases, and renal disorders. Recent advances in structural biology techniques have provided invaluable insights into the molecular mechanisms underlying ACE inhibition, facilitating the design and development of more effective therapeutic agents. This review focuses on the latest advancements in elucidating the structural basis for ACE inhibition. High-resolution crystallographic studies of minimally glycosylated individual domains of ACE have revealed intricate molecular details of the ACE catalytic N- and C-domains, and their detailed interactions with clinically relevant and newly designed domain-specific inhibitors. In addition, the recently elucidated structure of the glycosylated form of full-length ACE by cryo-electron microscopy (cryo-EM) has shed light on the mechanism of ACE dimerization and revealed continuous conformational changes which occur prior to ligand binding. In addition to these experimental techniques, computational approaches have also played a pivotal role in elucidating the structural basis for ACE inhibition. Molecular dynamics simulations and computational docking studies have provided atomic details of inhibitor binding kinetics and energetics, facilitating the rational design of novel ACE inhibitors with improved potency and selectivity. Furthermore, computational analysis of the motions observed by cryo-EM allowed the identification of allosteric binding sites on ACE. This affords new opportunities for the development of next-generation allosteric inhibitors with enhanced pharmacological properties. Overall, the insights highlighted in this review could enable the rational design of novel ACE inhibitors with improved efficacy and safety profiles, ultimately leading to better therapeutic outcomes for patients with hypertension and cardiovascular diseases.

Cholinesterases are well-known and widely studied enzymes crucial to human health and involved in neurology, Alzheimer\'s, and lipid metabolism. The protonation pattern of active sites of cholinesterases influences all the chemical processes within, including reaction, covalent inhibition by nerve agents, and reactivation. Despite its significance, our comprehension of the fine structure of cholinesterases remains limited. In this study, we employed enhanced-sampling quantum-mechanical/molecular-mechanical calculations to show that cholinesterases predominantly operate as dynamic mixtures of two protonation states. The proton transfer between two non-catalytic glutamate residues follows the Grotthuss mechanism facilitated by a mediator water molecule. We show that this uncovered complexity of active sites presents a challenge for classical molecular dynamics simulations and calls for special treatment. The calculated proton transfer barrier of 1.65 kcal/mol initiates a discussion on the potential existence of two coupled low-barrier hydrogen bonds in the inhibited form of butyrylcholinesterase. These findings expand our understanding of structural features expressed by highly evolved enzymes and guide future advances in cholinesterase-related protein and drug design studies.

The URH1p enzyme from the yeast Saccharomyces cerevisiae has gained significant interest due to its role in nitrogenous base metabolism, particularly involving uracil and nicotinamide salvage. Indeed, URH1p was initially classified as a nucleoside hydrolase (NH) with a pronounced preference for uridine substrate but was later shown to also participate in a Preiss-Handler-dependent pathway for recycling of both endogenous and exogenous nicotinamide riboside (NR) towards NAD+ synthesis. Here, we present the detailed enzymatic and structural characterisation of the yeast URH1p enzyme, a member of the group I NH family of enzymes. We show that the URH1p has similar catalytic efficiencies for hydrolysis of NR and uridine, advocating a dual role of the enzyme in both NAD+ synthesis and nucleobase salvage. We demonstrate that URH1p has a monomeric structure that is unprecedented for members of the NH homology group I, showing that oligomerisation is not strictly required for the N-ribosidic activity in this family of enzymes. The size, thermal stability and activity of URH1p towards the synthetic substrate 5-fluoruridine, a riboside precursor of the antitumoral drug 5-fluorouracil, make the enzyme an attractive tool to be employed in gene-directed enzyme-prodrug activation therapy against solid tumours.

RNA polymerases (RNAPs) carry out the first step in the central dogma of molecular biology by transcribing DNA into RNA. Despite their importance, much about how RNAPs work remains unclear, in part because the small (3.4 Angstrom) and fast (~40 ms/nt) steps during transcription were difficult to resolve. Here, we used high-resolution nanopore tweezers to observe the motion of single Escherichia coli RNAP molecules as it transcribes DNA ~1,000 times improved temporal resolution, resolving single-nucleotide and fractional-nucleotide steps of individual RNAPs at saturating nucleoside triphosphate concentrations. We analyzed RNAP during processive transcription elongation and sequence-dependent pausing at the yrbL elemental pause sequence. Each time RNAP encounters the yrbL elemental pause sequence, it rapidly interconverts between five translocational states, residing predominantly in a half-translocated state. The kinetics and force-dependence of this half-translocated state indicate it is a functional intermediate between pre- and post-translocated states. Using structural and kinetics data, we show that, in the half-translocated and post-translocated states, sequence-specific protein-DNA interaction occurs between RNAP and a guanine base at the downstream end of the transcription bubble (core recognition element). Kinetic data show that this interaction stabilizes the half-translocated and post-translocated states relative to the pre-translocated state. We develop a kinetic model for RNAP at the yrbL pause and discuss this in the context of key structural features.

Homologation of amino acids is the insertion or deletion of a methylene group to their side chain, which is a relatively uncommon chemical transformation observed in peptide natural product (NP) structure. Homologated amino acids can potentially make the NP more stable in a biological system, but its biosynthesis is yet to be understood. This study biochemically characterized the first of three unexplored enzymes in the homologation pathway of l-phenylalanine and l-tyrosine. Previously proposed reactions catalyzed by HphA were confirmed by reversed-phase high-performance liquid chromatography and tandem mass spectrometry analysis. The substrate profile and kinetic parameters showed high selectivity for the natural substrates and their close analogs. The comparability of HphA to homologous enzymes in primary metabolic pathways, 2-isopropylmate synthase and homocitrate synthase which are involved in l-leucine and l-lysine biosynthesis, respectively, was validated by bioinformatical and site-directed mutagenesis studies. The knowledge obtained from this study has deepened the understanding of the homologation of amino acids, which can lead to future combinatorial biosynthesis and metabolic engineering studies.

Members of the myosin superfamily of molecular motors are large mechanochemical ATPases that are implicated in an ever-expanding array of cellular functions. This review focuses on mammalian nonmuscle myosin-2 (NM2) paralogs, ubiquitous members of the myosin-2 family of filament-forming motors. Through the conversion of chemical energy into mechanical work, NM2 paralogs remodel and shape cells and tissues. This process is tightly controlled in time and space by numerous synergetic regulation mechanisms to meet cellular demands. We review how recent advances in structural biology together with elegant biophysical and cell biological approaches have contributed to our understanding of the shared and unique mechanisms of NM2 paralogs as they relate to their kinetics, regulation, assembly, and cellular function.

DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (-1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface \'swapping\' (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed \'swivelling\' mechanism for DNA gyrase (Gubaev et al., 2016).

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

Actin is a highly conserved and fundamental protein in eukaryotes and participates in a broad spectrum of cellular functions. Cells maintain a conserved ratio of actin isoforms, with muscle and non-muscle actins representing the main actin isoforms in muscle and non-muscle cells, respectively. Actin isoforms have specific and redundant functional roles and display different biochemistries, cellular localization, and interactions with myosins and actin-binding proteins. Understanding the specific roles of actin isoforms from the structural and functional perspective is crucial for elucidating the intricacies of cytoskeletal dynamics and regulation and their implications in health and disease. Here, we review how the structure contributes to the functional mechanisms of actin isoforms with a special emphasis on the questions of how post-translational modifications and disease-linked mutations affect actin isoforms biochemistry, function, and interaction with actin-binding proteins and myosin motors.