Mobile genetic elements (MGEs) drive evolution and adaptation throughout the tree of life. In bacteria, MGEs frequently transfer antibiotic resistance gene (ARGs) and are major drivers of resistance spreading. Their movements have been linked to the emergence of multidrug-resistant pathogens, including VRE, MRSA and ESBL, which present major public health challenges world-wide. Transposase enzymes that catalyze MGE movement, are the most abundant and most ubiquitous proteins in nature. Yet, their structure and biochemical mechanisms are poorly understood [1].

In this talk, I will present our recent discoveries on a group of transposases, which can effectively transfer ARG-carrying MGEs between diverse bacterial species in microbial communities. We have mapped the most wide-spread transposases in bacterial genomes [2] and reconstituted their molecular mechanisms [3, unpublished data]. We further characterized the biochemical steps of these MGEs and determined high-resolution crystal and cryo-EM structures of the protein-DNA assemblies involved in their transposition [4, unpublished data]. The results shed new light on the molecular strategies of transposase enzymes and elucidate how specific DNA structures enable these proteins to insert into diverse genomic sites, thus expanding ARG transfer. These insights open new possibilities for future strategies to block or prevent transposition and thus help control the spread of antibiotic resistance.

[1] Arinkin, V., Smyshlyaev, G. & Barabas, O. (2019) Curr Opin Struct Biol. 59, 168-177.

[2] Smyshlyaev, G., Bateman A. & Barabas O. (2021) Mol Syst Biol. e9880

[3] Lambertsen, L., Rubio-Cosials, A., Patil, K.R. & Barabas, O. (2018) Mol Microbiol. 107, 639-658.

[4] Rubio-Cosials, A., Schulz, E.C., Lambertsen, L., Smyshlyaev, G., Rojas-Cordova, C., Forslund, K., Karaca, E., Bebel, A., Bork, P. & Barabas, O. (2018) Cell 173, 208-220.

Recognition of DNA by proteins, both sequence and structure specific, is important in the functioning of the cell, such as in the processes of replication, transcription, and DNA repair. Twenty-five years after base flipping, a phenomenon whereby a base in normal B-DNA is swung completely out of the helix into an extrahelical position, was first observed in HhaI methyltransferase, we are still learning from and surprised by structures of protein-DNA complexes. The novel structures of the bacterium Caulobacter crescentus cell cycle-regulated DNA adenine methyltransferase (CcrM), as well as the newly discovered CamA enzyme (named for Clostridioides difficile adenine methyltransferase A) in complexes with double-stranded DNA containing their recognition sequence, will be discussed. Each of these enzymes affect their DNA substrate in a unique number of ways that are critical for their level of discrimination of their recognition DNA sequence.

CcrM in C.crescentus is responsible for maintenance methylation immediately after replication and methylates the adenine of hemimethylated GANTC. CcrM contains an N-terminal methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the five base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson–Crick polar hydrogen bonds to ensure high enzymatic specificity.

In the developed world, C. difficile is one of the leading causes of hospital-acquired infections. CamA-mediated methylation of the last adenine in CAAAAA is required for normal sporulation and biofilm production by this bacterium, a key step in disease transmission. Thus, selective inhibition of CamA has great therapeutic potential. CamA contains an N-terminal methyltransferase domain as well as a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These interactions provide sufficient sequence discrimination to ensure high specificity. In addition, this DNA methyltransferase has unusual features that may aide in discovery of a new selective antibiotic to combat C. difficile infection.

Knowledge acquired from these structures may also relate to other projects in our laboratory relating to mammalian epigenetics.

Enzymes with a protein kinase fold transfer phosphate from adenosine 5′-triphosphate (ATP) to substrates in a process known as phosphorylation. Here, we show that the Legionella meta-effector SidJ adopts a protein kinase fold, yet unexpectedly catalyzes protein polyglutamylation. SidJ is activated by host-cell calmodulin to polyglutamylate the SidE family of ubiquitin (Ub) ligases. Crystal structures of the SidJ-calmodulin complex reveal a protein kinase fold that catalyzes ATP-dependent isopeptide bond formation between the amino group of free glutamate and the gamma-carboxyl group of an active-site glutamate in SidE. We show that SidJ polyglutamylation of SidE, and the consequent inactivation of Ub ligase activity, is required for successful Legionella replication in a viable eukaryotic host cell. [1]

Here we also present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active site Glu in SidE resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE-ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation. [2]

[1] Black, M. H., Osinski, A., Gradowski, M., Servage, K. A., Pawłowski, K., Tomchick, D. R., Tagliabracci, V. S. (2019). Science 364, 787-792.

[2] Osinski, A., Black, M. H., Pawłowski, K., Chen, Z., Li, Y., Tagliabracci, V. S. (2021). bioRxiv doi: https://doi.org/10.1101/2021.04.13.439722

Results shown in this report are derived from work performed at the Structural Biology Center, Advanced Photon Source, Argonne National Laboratory. A portion of this research was supported by NIH grant U24GM129547 and performed at the Pacific Northwest Center for Cryo-EM at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. We thank the Structural Biology Laboratory and the Cryo Electron Microscopy Facility at UT Southwestern Medical Center which are partially supported by grant RP170644 from the Cancer Prevention & Research Institute of Texas (CPRIT) for cryo-EM studies.

Tick-borne encephalitis virus is the cause of tick-borne encephalitis, an important arboviral disease affecting population within European and north-eastern Asian countries. There is currently no specific treatment available although it is preventable by vaccination [1, 2]. The lack of specific antiviral together with low vaccination coverage allowed the expansion of the virus within the Europe in recent years.

In the lifecycle of TBEV, NS3 helicase domain plays an essential role in viral genome replication. This domain carries out three enzymatic activities: RNA 5’-triphosphatase, RNA helicase and ATP hydrolysis. The latter activity is coupled to and provides energy for the RNA helicase activity during unwinding of the double-stranded RNA replication intermediate [3]. To understand the coupling between ATP hydrolysis and NS3 helicase activity, we determined several crystal structures of NS3 helicase, either the apo form or in complex with non-hydrolyzable ATP-analogue (AMPPNP), ADP or ADP-Pi (post-hydrolysis state). These represent structural snapshots of the key stages in ATP hydrolysis and nucleotide exchange. We also demonstrated that the ATP hydrolysis is stimulated in the presence of ssRNA but not ssDNA, both of which bind but the latter acts as a competitive inhibitor. Thus, RNA selectivity is not due to specific binding but is encoded in the coupling mechanism.

The obtained structures served as basis for molecular dynamics simulations of NS3 helicase in complex with ssRNA. RNA binding in the post-hydrolysis state leads to an allosteric change which forces opening of the ATP binding site and allows release of the resulting inorganic phosphate ion, P_i. The allosteric change is commensurate with movement of ssRNA, suggesting that this step plays a key role in the tight coupling between helicase and ATPase activities.

Quinole-dependent nitric oxide reductases (qNORs), that use Nitric oxide (NO) to generate Nitrous oxide (N₂O) as the enzymatic product in agricultural and pathogenic conditions are of major importance to food production, environment and human health. These membrane-bound enzymes strongly contribute to environmental problem at the global level (N₂O is an ozone-depleting and greenhouse gas some 300-fold more potent than CO₂) and play significant roles in survival of pathogens (qNOR from human pathogenic bacterium, Neisseria meningitidis is responsible for detoxification of NO produced to combat immune response of the host). We have determined high-resolution cryo-EM structure of active quinol-dependent nitric oxide reductases (qNOR) from Neisseria meningitidis (Nm) and Alcaligenes xylosoxidans (Ax) at 3.06Å and 3.2Å, respectively [1,2]. For NmqNOR, we have also determined the crystallographic structure at 3.15Å. All of the crystallographic structures including that of NmqNOR are monomeric [3] while both cryoEM structures showed clear dimeric arrangement. We have identified a number of factors that may trigger destabilisation of helices necessary for preserving the integrity of dimer including the use of zinc in crystallisation. Activity assay of both NmqNOR and AxqNOR in the presence of ZnCl₂ or ZnSO₄ abolished the activity. A closer examination of the NmqNOR crystallographic and cryoEM structures revealed a significant movement of TMII where one of the Zn (called Zn1) is present in the crystallographic structure and the other was at Glu498 which is pulled away from binding to Fe_B in order to ligate the second Zn (called Zn2). It is unclear if the loss of activity is due to the binding of Zn1 located near TMII or is a consequence of the removal Glu498 from the coordination of Fe_B. The mutation of Glu to Ala led to an inactive enzyme with the size exclusion chromatography indicating the major species to be a monomer. We were able to determine the cryoEM structure of this monomer (~85kD) showing that the mutation, in addition to TMII movement, causes destabilisation of additional helices (TMIX and TMX). These results and their wider implications for structure determination of membrane proteins would be discussed in the context of enzyme mechanism.

Since resolution of the first macromolecular structure, the goal of structural biology has been to link structure to function. It is now widely accepted that the latter emerges from the structural dynamics animating the macromolecule, making characterization of intermediate (and sometime excited) states of high interest to further understand molecular processes and possibly control them. With the advent of serial crystallography at X-ray free electron lasers and synchrotrons, time-resolved crystallography, performed following a specific perturbation of the crystalline system (laser excitation, substrate soak, etc), is on the verge of becoming feasible on virtually all systems opening avenues to characterize such excited and/or intermediates states. Because crystallography is an ensemble-averaged method, however, an inherent limitation is that the occupancy of intermediate states must be high enough for the “probed state” under investigation to become visible in the electron density. This is generally not the case, with “perturbed” crystals rather existing as mixtures of initial and/or final state(s) with the “probed” state. Differences in structure factor amplitudes between the reference and “perturbed” dataset can allow calculation of Fourier difference maps (F_{obs,perturbed}-F_{obs,unperturbed}), in which only the differences between the states are depicted. An even more powerful approach is to generate extrapolated structure factor amplitudes (F_{extr,perturbed}) solely describing the intermediate state and and to use these to refine its structure using conventional refinement tools. Such data processing has in the past been performed by a handful of well-experienced crystallographers with strong knowledge of existing software but still remains out of reach for a wide audience.

Here, we will present a user-friendly program, Xtrapol8, written in python and exploiting the cctbx toolbox modules, that allows the calculation of high-quality Fourier difference maps, estimation of the occupancy of the intermediate state(s) in the crystals, and generation of extrapolated structure factor amplitudes. Briefly, the program uses Bayesian statistics to weight structure factor amplitude differences [1] which are then used to generate extrapolated structure factor amplitudes for a range of possible intermediate state occupancies, with distinct weighting schemes [2, 3] (Figure 1). Based on the comparison between experimental and calculated differences, i.e. solely on experimental observations, the correct occupancy of the intermediate state is determined and its structure refined, shedding light on conformational changes not visible before. With various user-controllable parameters of which defaults are carefully chosen, the program is adapted to be used by a wide audience of structural biologists, ranging from well-experienced crystallographers to newcomers in the field. We anticipate that this program will ease and accelerate the handling of time resolved structural data, and thereby the understanding of molecular processes underlying function in a variety of proteins.

[1] Ursby, T. & Bourgeois, D. (1997), Acta Crystallogr. Sect. A, 53, 564-575.

[2] Genick, U. .,Borgstahl, G. E., Ng, K., Ren, Z., Pradervand, C., Burke, P. M., Srajer, V., Teng, T. Y., Schildkamp, W., McRee, D. E., Moffat, K. & Getzoff, E. D. (1997), Science, 275, 1471-1475.

[3] Coquelle, N., Sliwa, M., Woodhouse, J., and others, Colletier, J.-P., Schlichting, I. & Weik, M. (2018), Nature chemistry, 10, 31–37.