Conformational dynamics is a fundamental aspect of enzymatic catalysis that, for example, can be linked to ligand binding and release, assembly of the active site, and the catalytic mechanism. The essential and metabolic enzyme adenylate kinase (AK) undergoes large-scale conformational changes in response to binding of its substrates ATP and AMP. As such, it has been intensely studied in search of linkages between dynamics and catalysis. For a complex conformational change to occur in a protein, whether it is of an induced fit or conformational selection nature, changes at several hinges are often required. Here, based on a comparative structure-function analysis of AK enzymes from E. coli and the archaea Odinarchaeota and from human AK1, we found that conformational changes in the enzymes are to a varying degree linked to bending, fraying, or unfolding/folding events of the termini of α-helices observed in various structural hot spots of the enzymes. The findings contribute with a mechanistic angle to how enzymatic dynamics and catalysis relate to the plasticity of the termini of α-helices.

The facultative pathogen Listeria monocytogenes uses a master regulator, PrfA, to tightly control the fitness- costly expression of its virulence factors. We found that PrfA activity is repressed via competitive occupancy of the binding site for the PrfA-activating cofactor, glutathione, by exogenous nutritional oligopeptides. The inhibitory peptides show different sequence and physicochemical properties, but how such a wide variety of oligopeptides can bind PrfA was unclear. Using crystal structure analysis of PrfA complexed with inhibitory tri- and tetrapeptides, we show here that the binding promiscuity is due to the ability of PrfA b5 in the glutathione-binding inter-domain tunnel to establish parallel or antiparallel b sheet-like interactions with the peptide backbone. Spacious tunnel pockets provide additional flexibility for unspecific peptide accommodation while providing selectivity for hydrophobic residues. Hydrophobic contributions from two adjacent peptide residues appear to be critical for efficient PrfA inhibitory binding. In contrast to glutathione, peptide binding prevents the conformational change required for the correct positioning of the DNA-binding helix-turn-helix motifs of PrfA, effectively inhibiting virulence gene expression.

Phosphoryl transfer is a fundamental reaction in cellular signaling and metabolism that requires Mg 2+ as an essential cofactor. While the primary function of Mg 2+ is electrostatic activation of substrates, such as ATP, the full spectrum of catalytic mechanisms exerted by Mg 2+ is not known. In this study, we integrate structural biology methods, molecular dynamic (MD) simulations, phylogeny, and enzymology assays to provide molecular insights into Mg 2+ -dependent structural reorganization in the active site of the metabolic enzyme adenylate kinase. Our results demonstrate that Mg 2+ induces a conformational rearrangement of the substrates (ATP and ADP), resulting in a 30° adjustment of the angle essential for reversible phosphoryl transfer, thereby optimizing it for catalysis. MD simulations revealed transitions between conformational substates that link the fluctuation of the angle to large-scale enzyme dynamics. The findings contribute detailed insight into Mg 2+ activation of enzymes and may be relevant for reversible and irreversible phosphoryl transfer reactions.

Bacteria resistant to one or several antibiotics are a very large problem, threatening not only treatment of infections but also surgery and cancer treatments. Thus, new types of antibacterial drugs are desperately needed.

Adenylate kinases play a crucial role in cellular energyhomeostasisthrough the interconversion of ATP, AMP, and ADP in all living organisms.Here, we explore how adenylate kinase (AdK) from Escherichiacoli interacts with diadenosine tetraphosphate (AP4A), aputative alarmone associated with transcriptional regulation, stress,and DNA damage response. From a combination of EPR and NMR spectroscopytogether with X-ray crystallography, we found that AdK interacts withAP4A with two distinct modes that occur on disparate time scales.First, AdK dynamically interconverts between open and closed stateswith equal weights in the presence of AP4A. On a much slower timescale, AdK hydrolyses AP4A, and we suggest that the dynamically accessedsubstrate-bound open AdK conformation enables this hydrolytic activity.The partitioning of the enzyme into open and closed states is discussedin relation to a recently proposed linkage between active site dynamicsand collective conformational dynamics.

Virulence factors enable pathogenic bacteria to infect host cells, establish infection, and contribute to disease progressions. In Gram-positive pathogens such as Staphylococcus aureus (Sa) and Enterococcus faecalis (Ef), the pleiotropic transcription factor CodY plays a key role in integrating metabolism and virulence factor expression. However, to date, the structural mechanisms of CodY activation and DNA recognition are not understood. Here, we report the crystal structures of CodY from Sa and Ef in their ligand-free form and their ligand-bound form complexed with DNA. Binding of the ligands-branched chain amino acids and GTP-induces conformational changes in the form of helical shifts that propagate to the homodimer interface and reorient the linker helices and DNA binding domains. DNA binding is mediated by a non-canonical recognition mechanism dictated by DNA shape readout. Furthermore, two CodY dimers bind to two overlapping binding sites in a highly cooperative manner facilitated by cross-dimer interactions and minor groove deformation. Our structural and biochemical data explain how CodY can bind a wide range of substrates, a hallmark of many pleiotropic transcription factors. These data contribute to a better understanding of the mechanisms underlying virulence activation in important human pathogens.

Enzymatic catalysis is critically dependent on selectivity, active site architecture, and dynamics. To contribute insights into the interplay of these properties, we established an approach with NMR, crystallography, and MD simulations focused on the ubiquitous phosphotransferase adenylate kinase (AK) isolated from Odinarchaeota (OdinAK). Odinarchaeota belongs to the Asgard archaeal phylum that is believed to be the closest known ancestor to eukaryotes. We show that OdinAK is a hyperthermophilic trimer that, contrary to other AK family members, can use all NTPs for its phosphorylation reaction. Crystallographic structures of OdinAK-NTP complexes revealed a universal NTP-binding motif, while 19 F NMR experiments uncovered a conserved and rate-limiting dynamic signature. As a consequence of trimerization, the active site of OdinAK was found to be lacking a critical catalytic residue and is therefore considered to be “atypical.” On the basis of discovered relationships with human monomeric homologs, our findings are discussed in terms of evolution of enzymatic substrate specificity and cold adaptation.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

Amyloid transthyretin (ATTR) amyloidosis is characterized by the abnormal accumulation of ATTR fibrils in multiple organs. However, the structure of ATTR fibrils from the eye is poorly understood. Here, we used cryo-EM to structurally characterize vitreous body ATTR fibrils. These structures were distinct from previously characterized heart fibrils, even though both have the same mutation and type A pathology. Differences were observed at several structural levels: in both the number and arrangement of protofilaments, and the conformation of the protein fibril in each layer of protofilaments. Thus, our results show that ATTR protein structure and its assembly into protofilaments in the type A fibrils can vary between patients carrying the same mutation. By analyzing and matching the interfaces between the amino acids in the ATTR fibril with those in the natively folded TTR, we are able to propose a mechanism for the structural conversion of TTR into a fibrillar form.