RNA viruses such as coronaviruses, flaviviruses, and henipaviruses represent major international health threats. Whilst these viruses replicate in the cytoplasm, they encode accessory proteins that target the host nuclear transport machinery to suppress innate immune pathways. Specifically, these virus proteins target the nuclear import receptor importin-a (IMPa) and inhibit host immune responses from entering the nucleus and triggering interferon (IFN) release. This immune evasion strategy is a critical component of virus pathogenicity, yet details of these interactions (including mechanism(s) of binding specificity with IMPa isoforms) remain unresolved. Here we describe the interfaces between these viral immune regulatory proteins and specific IMPA host receptors as targets for development of novel antivirals.

Prevalence of drug-resistant strains of causative agents of age old diseases pneumonia and tuberculosis (TB), has urged focus on exploring novel targets and development of new therapeutics with a fresh perspective in the battle against antibiotic resistance. Now-a-days bioactive compounds from natural origin are superseding the use of synthetic compounds due to structural and chemical diversity [1]. Our research illustrates the power of integrative structural biology in the discovery of inhibitors against two potential drug targets - (1) Serine acetyltransferase (also known as CysE), an enzyme of de novo cysteine biosynthetic pathway, and (2) Isocitrate lyases with role in both glyoxylate cycle and methylcitrate cycle

(1) CysE catalyzes the production of O-acetyl-L-serine (OAS) from acetyl-CoA and L-serine. The enzyme, essential for survival in a mouse model of TB infection [2], is absent in Homo sapiens. Therefore, this target is worth exploring for developing new antimicrobial compounds. The crystal structure of K. pneumoniae (Kpn) CysE was solved and used as a receptor for blind docking of natural compounds with documented antioxidant, antibacterial, respiratory stimulant, anti-inflammatory, and bronco-dilatory activities. L-Cys, a feedback inhibitor of CysE which binds at the active site was also docked as a positive control (Fig.1a). The best binders were tested for the inhibitory potential of CysE and quercetin was identified as the most potent inhibitor (Fig. 1b). MD simulations verified it as an allosteric inhibitor that binds at the trimer-trimer interface distal to the active and cofactor binding site.

(2) Isocitrate lyases (ICL1/ICL2) are essential for persistence of M. tuberculosis (Mtb) in its host [3] as they play an important role in metabolism of even and odd chain fatty acids via β-oxidation. Though high resolution crystal structures of Mtb ICL1 are available in PDB since 2000, and GlaxoSmithKline-TB Alliance launched high throughput screening of 900,000 compounds to identify ICL1 inhibitor, their efforts culminated in modest succes, in view of poor characterization of ICL2 structure-function relationship. We purified both Rv1915 and Rv1916 and characterized them possessing dual isocitrate and methylisocitrate lyase activities akin to ICL1[4]. In silico screening of natural compounds has yielded an inhibitor which is able to abolish both the activities in all Mtb ICLs.

[1]. Pereira D. M., Andrade C., Valentão P., & Andrade P. B. (2017). “Natural Products Targeting Clinically Relevant Enzymes, pp. 1–18. Wiley-VCH Verlag GmbH & Co. KGaA,

[2]. Sassetti C. M. & Rubin E. J. (2003).Proc. Natl. Acad. Sci. USA 100:12989-94

[3]. McKinney J. D., zu Bentrup K. H., Muñoz-Elías E. J., et al (2000). Nature 406:735–738.

[4]. Gould T. A., van de Langemheen H., Munoz-Elias E. J., et al (2006). Mol Microbiol 61:940–947.

Metal ions are essential for all forms of life. In prokaryotes, ATP-binding cassette (ABC) permeases serve as the primary import pathway for many micronutrients including the first-row transition metal manganese. However, the structural features of ionic metal transporting ABC permeases have remained undefined. This presentation will describe the crystal structure of the manganese transporter PsaBC from Streptococcus pneumoniae in an open-inward conformation. The Type II transporter has a tightly closed transmembrane channel due to ‘extracellular gating’ residues that prevent water permeation or ion reflux. Below these residues, the channel contains a hitherto unreported metal coordination site, which is essential for manganese translocation. These structural features are highly conserved in metal-specific ABC transporters and are represented throughout the kingdoms of life. Collectively, our results define the structure of PsaBC and reveal the features required for divalent cation transport.

We know so little about how bacteria utilise surface virulence factors to colonise, infect, persist and cause disease in their hosts. The largest group of these virulence factors are the autotransporters, where although they employ a simple process for translocation to the bacterial surface, their functional passenger domains show a diverse range of pathogenic functions such as promoting adhesion, biofilm formation, invasion and tissue destruction. Despite extensive international efforts at the genotype-phenotype level that have confirmed the association of autotransporters with bacterial pathogenesis, less than 0.6 % of their structures have been determined with very little information on their molecular mechanisms of action.

With 10 new structures of autotransporter passenger domains over the past few years our group has been leading this area of research. Taking advantage of many autotransporter passenger domains being based upon large >500 residue β-solenoid structures, we have successfully employed Xenon derivatisation at the Australian Synchrotron to acquire anomalous signal for structure determination by single isomorphous replacement. More importantly, we have used our crystal structures to inform a comprehensive array of biophysical, biochemical and microbiological approaches to uncover the mode of action of the autotransporters and their roles in bacterial pathogenesis. Using this approach we were the first to determine the molecular mechanism of an autotransporter adhesin¹. We found that this Ag43 adhesin from Uropathogenic E. coli (UPEC) promoted bacterial biofilms through a self-association mechanism between neighbouring E. coli cell surfaces. This knowledge on biofilms is critical given their contribution to bacterial chronic infections and the development of antibiotic resistance.

Here we present the first crystal structure and mechanism of action of an autotransporter adhesin that binds to host tissue to facilitate bacterial colonisation². The crystal structure of UpaB from UPEC was found to display significant modifications to its β-helix that creates two different binding sites, allowing it to interact simultaneously with both host surface proteins and polysaccharides. As shown in live animal models, both sites co-operate to achieve bacterial colonisation. In contrast to Ag43 that forms self-associations that lead to biofilms, UpaB through directly binding host factors to facilitate colonisation creates a second mechanistic group of the autotransporter adhesins.

Returning to Ag43, we also investigate the conservation of its self-association mechanism with 3 new crystal structures of Ag43 homologues from widespread E. coli pathogens³. We show that adaptations to this mechanism of action alter the kinetics of bacterial aggregation and biofilm formation, presumably to suit the different E. coli pathogens to their specific infection sites. Even more importantly, we are using our molecular knowledge on autotransporters such as Ag43 to develop new classes of anti-bacterial inhibitors. To date we have developed and patented a successful inhibitor that targets Ag43 to prevent pathogenic E. coli biofilms⁴. Again using X-ray crystallography we have determined the structure of the first autotransporter adhesin-inhibitor complex to fully understand how this novel inhibitor interacts with Ag43 and blocks its funtion.

Figure 1A: Ag43 self-associates between E. coli surfaces to promote aggregation and biofilm formation. B. UpaB directly binds both host proteins and carbohydrates to promote UPEC colonisation.

[1] Heras B, Totsika M, Peters KM, Paxman JJ, Gee CL, Jarrott RJ, Perugini MA, Whitten AE and Schembri MA (2014). Proc Natl Acad Sci USA 111, 457-462.

[2] Paxman JJ, Lo A, Sullivan MJ, Panjikar S, Kuiper M, Whitten AE, Wang G, Luan CH, Moriel DG, Tan L, Peters KM, Gee C, Ulett GC, Schembri MA and Heras B. (2019). Nat. Commun. Apr 29;10(1);1967.

[3] Vo J, Martínez Ortiz GC, Totsika M, Lo A, Whitten AE, Hor L, Peters KM, Ageorges V, Caccia N, Desvaux M, Schembri M, Paxman JJ and Heras B (2021). ELife (under revision).
[4] Heras B, Paxman JJ, Schembri M and Lo A (2019) International Patent (PCT/AU2019/050893).

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The evolutionarily conserved Retromer complex (Vps35-Vps26-Vps29) is a master regulator responsible for endosomal membrane trafficking and signalling. It is known that mutations in Retromer can cause late-onset Parkinson’s disease, and can also be hijacked by viral and bacterial pathogens during cellular infection. Seeking tools to modulate Retromer function would provide new avenues in understanding its function and the associated diseases. Here we employed the random nonstandard peptides integrated discovery (RaPID) approach to identify a group of macrocyclic peptides capable of binding to Retromer with high affinity. Our crystal structures show that five of the macrocyclic peptides bind to human Vps29 via a di-peptide Pro-Leu sequence. Interestingly, these peptides structurally mimic known interacting proteins including TBC1D5, VARP, and the bacterial effector RidL, and potently inhibit their interaction with Retromer in vitro and in cells. In addition, we found that these Vps29-binding macrocyclic peptides also mimic the binding between thermophilic yeast Vps29 and the unstructured N-terminal domain of Vps5. Disruption of this previously uncharacterized interaction by macrocyclic peptides negatively affect yeast Retromer, Vps5 and Vps17 to form stable heteropentameric complex. By contrast, mutagenesis and cryoEM show that macrocyclic peptide RT-L4 binds Retromer at the Vps35 and Vps26 interface, and it can act as a molecular chaperone to stabilise the complex with minimal disruptive effects on Retromer’s ability to interact with its accessory proteins. Finally, using reversible cell permeabilization approach, we demonstrate that both the Retromer inhibiting and stabilizing macrocyclic peptides can specifically co-label Vps35-positive endosomal structures, and can be used as baits for purifying Retromer from cells and subsequent proteomic analyses. We believe these macrocyclic peptides can be used as a novel toolbox for the study of Retromer-mediated endosomal trafficking, and sheds light on developing novel therapeutic modifiers of Retromer function.

The class D serine β-lactamases comprise a superfamily of almost 900 enzymes capable of conferring high-level resistance to β-lactam antibiotics, predominantly the penicillins including oxacillin (Fig. 1) and cloxacillin, and some early generation cephalosporins. In recent years it has been discovered that some members of the class D β-lactamase superfamily have evolved the ability to deactivate carbapenems (Fig. 1), last resort β-lactam antibiotics generally held in reserve for highly drug resistant bacterial infections. These enzymes are collectively known as Carbapenem-Hydrolyzing Class D serine β-Lactamases or CHDLs [1,2]. Most alarmingly, a large number (>500) of these CHDLs have appeared in several Acinetobacter baumannii strains, leading the CDC to elevate this once nosocomial infection of little clinical importance into a major opportunistic pathogen, now deemed to be an urgent global threat [3] with mortality rates from infections by resistant strains often exceeding 50% [4].

The mechanism of β-lactam deactivation by the class D serine β-lactamases involves the covalent binding of the antibiotic to an active site serine to form an acyl-enzyme intermediate (acylation). This is followed by hydrolysis of the acyl bond (deacylation), catalysed by a water molecule activated by a carboxylated lysine residue [5]. It was initially thought that the carbapenems acted as potent inhibitors of the class D enzymes since formation of the covalent acyl-enzyme intermediate expelled all water molecules from the active site, and stereochemistry of the side group at carbon 6 of the β-lactam ring effectively blocked access into the pocket housing the catalytic lysine, thus preventing the deacylation step. Our recent structural studies on three CHDLs (OXA-23, OXA-48 and OXA-143) [4,6,7] have indicated that their carbapenem hydrolysing ability may be due to small-scale dynamics of two surface hydrophobic residues which form a hydrophobic lid over the internal pocket housing the catalytic lysine. Movement of one or both of these residues allow for the transient opening and closing of a channel (Fig. 2) through which water molecules from the milieu can enter the lysine pocket to facilitate the deacylation reaction. Although the hydrophobic residues responsible for the channel formation are present in all class D β-lactamases, sequence and structural differences nearby may be responsible for the evolution of carbapenemase activity in the CHDLs. Current and future work aimed at non-covalent inhibitor development in OXA-23, and improved covalent inhibitor design focused on blocking access to the catalytic lysine pocket in OXA-23 and OXA-48 will be presented.

[1] Queenan A.M. & Bush K. (2007). Clin. Microbiol. Rev. 20:440.

[2] Walther-Rasmussen J. & Hoiby N. (2006). J. Antimicrob. Chemother. 57:373.

[3] https://www.cdc.gov/drugresistance/biggest-threats.html

[4] Smith C.A., Antunes N.T., Stewart N.K., Toth M., Kumarasiri M., Chang M., Mobashery S. & Vakulenko S.B. (2013). Chem. Biol. 20:1107.

[5] Golemi D., Maveyraud L., Vakulenko S., Samama J.P. & Mobashery S. (2001). Proc. Natl. Acad. Sci. 98:14280.

[6] Toth M., Smith C.A., Antunes N.T., Stewart N.K., Maltz L. & Vakulenko S.B. (2017). Acta. Crystallogr. D73:692.

[7] Smith C.A., Stewart N.K., Toth M. & Vakulenko S.B. (2019). Antimicrob. Agents Chemother. 63:e01202-19.