Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli.

Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY" segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

Disulfide Bonds Play a Critical Role in the Structure and Function of the Receptor-binding Domain of the SARS-CoV-2 Spike Antigen.

The current coronavirus pandemic is exerting a tremendously detrimental impact on global health. The Spike proteins of coronaviruses, responsible for cell receptor binding and viral internalization, possess multiple and frequently conserved disulfide bonds raising the question about their role in these proteins. Here, we present a detailed structural and functional investigation of the disulfide bonds of the SARS-CoV-2 Spike receptor-binding domain (RBD). Molecular dynamics simulations of the RBD predict increased flexibility of the surface loops when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the disulfide bond-containing surface loop (residues 456-490) that participates in the formation of the interaction surface with the Spike cell receptor ACE2. In vitro, disulfide bond reducing agents affect the RBD secondary structure, lower its melting temperature from 52 °C to 36-39 °C and decrease its binding affinity to ACE2 by two orders of magnitude at 37 °C. Consistent with these in vitro findings, the reducing agents tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) were able to inhibit viral replication at low millimolar levels in cell-based assays. Our research demonstrates the mechanism by which the disulfide bonds contribute to the molecular structure of the RBD of the Spike protein, allowing the RBD to execute its viral function.

The Legionella kinase LegK7 exploits the Hippo pathway scaffold protein MOB1A for allostery and substrate phosphorylation.

During infection, the bacterial pathogen Legionella pneumophila manipulates a variety of host cell signaling pathways, including the Hippo pathway which controls cell proliferation and differentiation in eukaryotes. Our previous studies revealed that L. pneumophila encodes the effector kinase LegK7 which phosphorylates MOB1A, a highly conserved scaffold protein of the Hippo pathway. Here, we show that MOB1A, in addition to being a substrate of LegK7, also functions as an allosteric activator of its kinase activity. A crystallographic analysis of the LegK7-MOB1A complex revealed that the N-terminal half of LegK7 is structurally similar to eukaryotic protein kinases, and that MOB1A directly binds to the LegK7 kinase domain. Substitution of interface residues critical for complex formation abrogated allosteric activation of LegK7 both in vitro and within cells and diminished MOB1A phosphorylation. Importantly, the N-terminal extension (NTE) of MOB1A not only regulated complex formation with LegK7 but also served as a docking site for downstream substrates such as the transcriptional coregulator YAP1. Deletion of the NTE from MOB1A or addition of NTE peptides as binding competitors attenuated YAP1 recruitment to and phosphorylation by LegK7. By providing mechanistic insight into the formation and regulation of the LegK7-MOB1A complex, our study unravels a sophisticated molecular mimicry strategy that is used by L. pneumophila to take control of the host cell Hippo pathway.

Regulation of Shigella Effector Kinase OspG through Modulation of Its Dynamic Properties.

Gram-negative pathogens secrete effector proteins into human cells to modulate normal cellular processes and establish a bacterial replication niche. Shigella and pathogenic Escherichia coli possess homologous effector kinases, OspG and NleH1/2, respectively. Upon translocation, OspG but not NleH binds to ubiquitin and a subset of E2~Ub conjugates, which was shown to activate its kinase activity. Here we show that OspG, having a minimal kinase fold, acquired a novel mechanism of regulation of its activity. Binding of the E2~Ub conjugate to OspG not only stimulates its kinase activity but also increases its optimal temperature for activity to match the human body temperature and stabilizes its labile C-terminal domain. The melting temperature (Tm) of OspG alone is only 31 °C, as compared to 41 °C to NleH1/2 homologs. In the presence of E2~Ub, the Tm of OspG increases to ~42 °C, while Ub by itself increases the Tm to 39 °C. Moreover, OspG alone displays maximal activity at 26 °C, while in the presence of E2~Ub, maximal activity occurs at ~42 °C. Using NMR and molecular dynamics calculations, we have identified the C-terminal lobe and, in particular, the C-terminal helix, as the key elements responsible for lower thermal stability of OspG as compared to homologous effector kinases.

Structural Organization of Enzymes of the Phenylacetate Catabolic Hybrid Pathway.

Aromatic compounds are the second most abundant class of molecules on the earth and frequent environmental pollutants. They are difficult to metabolize due to an inert chemical structure, and of all living organisms, only microbes have evolved biochemical pathways that can open an aromatic ring and catabolize thus formed organic molecules. In bacterial genomes, the phenylacetate (PA) utilization pathway is abundant and represents the central route for degradation of a variety of organic compounds, whose degradation reactions converge at this pathway. The PA pathway is a hybrid pathway and combines the dual features of aerobic metabolism, i.e., usage of both oxygen to open the aromatic ring and of anaerobic metabolism-coenzyme A derivatization of PA. This allows the degradation process to be adapted to fluctuating oxygen conditions. In this review we focus on the structural and functional aspects of enzymes and their complexes involved in the PA degradation by the catabolic hybrid pathway. We discuss the ability of the central PaaABCE monooxygenase to reversibly oxygenate PA, the controlling mechanisms of epoxide concentration by the pathway enzymes, and the similarity of the PA utilization pathway to the benzoate utilization Box pathway and ß-oxidation of fatty acids.

Structural insight into effector proteins of Gram-negative bacterial pathogens that modulate the phosphoproteome of their host.

Invading pathogens manipulate cellular process of the host cell to establish a safe replicative niche. To this end they secrete a spectrum of proteins called effectors that modify cellular environment through a variety of mechanisms. One of the most important mechanisms is the manipulation of cellular signaling through modifications of the cellular phosphoproteome. Phosphorylation/dephosphorylation plays a pivotal role in eukaryotic cell signaling, with ∼ 500 different kinases and ∼ 130 phosphatases in the human genome. Pathogens affect the phosphoproteome either directly through the action of bacterial effectors, and/or indirectly through downstream effects of host proteins modified by the effectors. Here we review the current knowledge of the structure, catalytic mechanism and function of bacterial effectors that modify directly the phosphorylation state of host proteins. These effectors belong to four enzyme classes: kinases, phosphatases, phospholyases and serine/threonine acetylases.

Structural basis for the inhibition of host protein ubiquitination by Shigella effector kinase OspG.

Shigella invasion of its human host is assisted by T3SS-delivered effector proteins. The OspG effector kinase binds ubiquitin and ubiquitin-loaded E2-conjugating enzymes, including UbcH5b and UbcH7, and attenuates the host innate immune NF-kB signaling. We present the structure of OspG bound to the UbcH7∼Ub conjugate. OspG has a minimal kinase fold lacking the activation loop of regulatory kinases. UbcH7∼Ub binds OspG at sites remote from the kinase active site, yet increases its kinase activity. The ubiquitin is positioned in the "open" conformation with respect to UbcH7 using its I44 patch to interact with the C terminus of OspG. UbcH7 binds to OspG using two conserved loops essential for E3 ligase recruitment. The interaction of the UbcH7∼Ub with OspG is remarkably similar to the interaction of an E2∼Ub with a HECT E3 ligase. OspG interferes with the interaction of UbcH7 with the E3 parkin and inhibits the activity of the E3.

NleH defines a new family of bacterial effector kinases.

Upon host cell infection, pathogenic Escherichia coli hijacks host cellular processes with the help of 20-60 secreted effector proteins that subvert cellular processes to create an environment conducive to bacterial survival. The NleH effector kinases manipulate the NF-κB pathway and prevent apoptosis. They show low sequence similarity to human regulatory kinases and contain two domains, the N-terminal, likely intrinsically unfolded, and a C-terminal kinase-like domain. We show that these effectors autophosphorylate on sites located predominantly in the N-terminal segment. The kinase domain displays a minimal kinase fold, but lacks an activation loop and the GHI subdomain. Nevertheless, all catalytically important residues are conserved. ATP binding proceeds with minimal structural rearrangements. The NleH structure is the first for the bacterial effector kinases family. NleHs and their homologous effector kinases form a new kinase family within the cluster of eukaryotic-like kinases that includes also Rio, Bud32, and KdoK families.

Family of phenylacetyl-CoA monooxygenases differs in subunit organization from other monooxygenases.

The phenylacetate degradation pathway is present in a wide range of microbes. A key component of this pathway is the four-subunit phenylacetyl-coenzyme A monooxygenase complex (PA-CoA MO, PaaACBE) that catalyzes the insertion of an oxygen in the aromatic ring of PA. This multicomponent enzyme represents a new family of monooxygenases. We have previously determined the structure of the PaaAC subcomplex of catalytic (A) and structural (C) subunits and shown that PaaACB form a stable complex. The PaaB subunit is unrelated to the small subunits of homologous monooxygenases and its role and organization of the PaaACB complex is unknown. From low-resolution crystal structure, electron microscopy and small angle X-ray scattering we show that the PaaACB complex forms heterohexamers, with a homodimer of PaaB bridging two PaaAC heterodimers. Modeling the interactions of reductase subunit PaaE with PaaACB suggested that a unique and conserved 'lysine bridge' constellation near the Fe-binding site in the PaaA subunit (Lys68, Glu49, Glu72 and Asp126) may form part of the electron transfer path from PaaE to the iron center. The crystal structure of the PaaA(K68Q/E49Q)-PaaC is very similar to the wild-type enzyme structure, but when combined with the PaaE subunit the mutant showed 20-50 times reduced activity, supporting the functional importance of the 'lysine bridge'.

Protein-protein interactions in the ß-oxidation part of the phenylacetate utilization pathway: crystal structure of the PaaF-PaaG hydratase-isomerase complex.

Microbial anaerobic and so-called hybrid pathways for degradation of aromatic compounds contain ß-oxidation-like steps. These reactions convert the product of the opening of the aromatic ring to common metabolites. The hybrid phenylacetate degradation pathway is encoded in Escherichia coli by the paa operon containing genes for 10 enzymes. Previously, we have analyzed protein-protein interactions among the enzymes catalyzing the initial oxidation steps in the paa pathway (Grishin, A. M., Ajamian, E., Tao, L., Zhang, L., Menard, R., and Cygler, M. (2011) J. Biol. Chem. 286, 10735-10743). Here we report characterization of interactions between the remaining enzymes of this pathway and show another stable complex, PaaFG, an enoyl-CoA hydratase and enoyl-Coa isomerase, both belonging to the crotonase superfamily. These steps are biochemically similar to the well studied fatty acid ß-oxidation, which can be catalyzed by individual monofunctional enzymes, multifunctional enzymes comprising several domains, or enzymatic complexes such as the bacterial fatty acid ß-oxidation complex. We have determined the structure of the PaaFG complex and determined that although individually PaaF and PaaG are similar to enzymes from the fatty acid ß-oxidation pathway, the structure of the complex is dissimilar from bacterial fatty acid ß-oxidation complexes. The PaaFG complex has a four-layered structure composed of homotrimeric discs of PaaF and PaaG. The active sites of PaaF and PaaG are adapted to accept the intermediary components of the Paa pathway, different from those of the fatty acid ß-oxidation. The association of PaaF and PaaG into a stable complex might serve to speed up the steps of the pathway following the conversion of phenylacetyl-CoA to a toxic and unstable epoxide-CoA by PaaABCE monooxygenase.

Structural and functional studies of the Escherichia coli phenylacetyl-CoA monooxygenase complex.

The utilization of phenylacetic acid (PA) in Escherichia coli occurs through a hybrid pathway that shows features of both aerobic and anaerobic metabolism. Oxygenation of the aromatic ring is performed by a multisubunit phenylacetyl-coenzyme A oxygenase complex that shares remote homology of two subunits to well studied bacterial multicomponent monooxygenases and was postulated to form a new bacterial multicomponent monooxygenase subfamily. We expressed the subunits PaaA, B, C, D, and E of the PA-CoA oxygenase and showed that PaaABC, PaaAC, and PaaBC form stable subcomplexes that can be purified. In vitro reconstitution of the oxygenase subunits showed that each of the PaaA, B, C, and E subunits are necessary for catalysis, whereas PaaD is not essential. We have determined the crystal structure of the PaaAC complex in a ligand-free form and with several CoA derivatives. We conclude that PaaAC forms a catalytic core with a monooxygenase fold with PaaA being the catalytic α subunit and PaaC, the structural ß subunit. PaaAC forms heterotetramers that are organized very differently from other known multisubunit monooxygenases and lacks their conservative network of hydrogen bonds between the di-iron center and protein surface, suggesting different association with the reductase and different mechanisms of electron transport. The PaaA structure shows adaptation of the common access route to the active site for binding a CoA-bound substrate. The enzyme-substrate complex shows the orientation of the aromatic ring, which is poised for oxygenation at the ortho-position, in accordance with the expected chemistry. The PA-CoA oxygenase complex serves as a paradigm for the new subfamily multicomponent monooxygenases comprising several hundred homologs.

Crystallization and preliminary X-ray analysis of PaaAC, the main component of the hydroxylase of the Escherichia coli phenylacetyl-coenzyme A oxygenase complex.

The Escherichia coli paa operon encodes enzymes of the phenylacetic acid-utilization pathway that metabolizes phenylacetate in the form of a coenzyme A (CoA) derivative. The phenylacetyl-coenzyme A oxygenase complex, which has been postulated to contain five components designated PaaABCDE, catalyzes ring hydroxylation of phenylacetyl-CoA. The PaaAC subcomplex shows low sequence similarity to other bacterial multicomponent monooxygenases (BMMs) and forms a separate branch on the phylogenetic tree. PaaAC, which catalyzes the hydroxylation reaction, was purified and crystallized in the absence of a bound ligand as well as in complexes with CoA, 3-hydroxybutyryl-CoA, benzoyl-CoA and the true substrate phenylacetyl-CoA. Crystals of the ligand-free enzyme belonged to space group P2(1)2(1)2(1) and diffracted to 2.65 A resolution, whereas complexes with CoA and its derivatives crystallized in space group P4(1)2(1)2 and diffracted to approximately 2.0 A resolution. PaaAC represents the first crystallized BMM hydroxylase that utilizes a CoA-linked substrate.