Structural basis for proenzyme maturation, substrate recognition, and ligation by a hyperactive peptide asparaginyl ligase.

Peptide ligases are versatile enzymes that can be utilized for precise protein conjugation for bioengineering applications. Hyperactive peptide asparaginyl ligases (PALs), such as butelase-1, belong to a small class of enzymes from cyclotide-producing plants that can perform site-specific, rapid ligation reactions after a target peptide asparagine/aspartic acid (Asx) residue binds to the active site of the ligase. How PALs specifically recognize their polypeptide substrates has remained elusive, especially at the prime binding side of the enzyme. Here we report crystal structures that capture VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzyme's S1 pocket and why a hydrophobic residue is required at the P2' position. Besides illustrating the anchoring role played by P1 and P2' residues, these results uncover a role for the Gatekeeper residue at the surface of the S2 pocket in shifting the nonprime portion of the substrate and, as a result, the activity toward ligation or hydrolysis. These results suggest a picture for proenzyme maturation in the vacuole and will inform the rational design of peptide ligases with tailored specificities.

Crystal structure and functional analysis of mycobacterial erythromycin resistance methyltransferase Erm38 reveals its RNA-binding site.

Erythromycin resistance methyltransferases (Erms) confer resistance to macrolide, lincosamide, and streptogramin antibiotics in Gram-positive bacteria and mycobacteria. Although structural information for ErmAM, ErmC, and ErmE exists from Gram-positive bacteria, little is known about the Erms in mycobacteria, as there are limited biochemical data and no structures available. Here, we present crystal structures of Erm38 from Mycobacterium smegmatis in apoprotein and cofactor-bound forms. Based on structural analysis and mutagenesis, we identified several catalytically critical, positively charged residues at a putative RNA-binding site. We found that mutation of any of these sites is sufficient to abolish methylation activity, whereas the corresponding RNA-binding affinity of Erm38 remains unchanged. The methylation reaction thus appears to require a precise ensemble of amino acids to accurately position the RNA substrate, such that the target nucleotide can be methylated. In addition, we computationally constructed a model of Erm38 in complex with a 32-mer RNA substrate. This model shows the RNA substrate stably bound to Erm38 by a patch of positively charged residues. Furthermore, a π-π stacking interaction between a key aromatic residue of Erm38 and a target adenine of the RNA substrate forms a critical interaction needed for methylation. Taken together, these data provide valuable insights into Erm-RNA interactions, which will aid subsequent structure-based drug design efforts.

Structural determinants for peptide-bond formation by asparaginyl ligases.

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1' sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs.

Cutibacterium acnes: Much ado about maybe nothing much.

Cutibacterium acnes (also known as Propionibacterium acnes) has long been implicated in the pathogenesis of acne, inspiring both therapeutic and personal care approaches aiming to control the disease by controlling the bacterium. The purported association has made people with acne feel dirty and led to the-at times excessive-use of cleansers, antiseptics and antibiotics for the condition. However, recent evidence seems to weaken the case for C. acnes' involvement. New genetics and molecular biology findings strongly suggest that abnormal differentiation of sebaceous progenitor cells causes comedones, the primary lesions in acne. Comodegenesis is initiated by androgens and is unlikely to be triggered by C. acnes, which probably doesn't affect sebaceous differentiation. Is there still a place for it in this understanding of acne? It is necessary to critically address this question because it has consequences for treatment. Antibiotic use for acne noticeably contributes to microbial drug resistance, which we can ill afford. In this Viewpoint, we explore if and how C. acnes (still) fits into the developing view on acne. We also briefly discuss the implications for therapy in the light of antibiotic resistance and the need for more targeted therapies.

Crystal structure of the periplasmic sensor domain of histidine kinase VbrK suggests indirect sensing of ß-lactam antibiotics.

Bacterial two-component regulatory systems (TCS) play important roles in sensing environmental stimuli and responding to them by regulating gene expression. VbrK/VbrR, a TCS in Vibrio parahaemolyticus, confers resistance to ß-lactam antibiotics through activating a ß-lactamase gene. Its periplasmic sensor domain was previously suggested to detect ß-lactam antibiotics by direct binding. Here, we report a crystal structure of the periplasmic sensing domain of VbrK (VbrKSD) using sulfur-based single-wavelength anomalous diffraction (S-SAD) phasing. Contrary to most bacterial sensor domains which form dimers, we show that VbrKSD is a monomer using size exclusion chromatography coupled with multi-angle light scattering. This observation is also supported by molecular dynamics simulations. To quantify the binding affinity of ß-lactam antibiotics to VbrKSD, we performed isothermal titration calorimetry and other biophysical analyses. Unexpectedly, VbrKSD did not show any significant binding to ß-lactam antibiotics. Therefore, we propose that the detection of ß-lactam antibiotics by VbrK is likely to be indirect via an as yet unidentified mechanism.

Structural Mimicry of the Dengue Virus Envelope Glycoprotein Revealed by the Crystallographic Study of an Idiotype-Anti-idiotype Fab Complex.

A detailed understanding of the fine specificity of serotype-specific human antibodies is vital for the development and evaluation of new vaccines for pathogenic flaviviruses such as dengue virus (DENV) and Zika virus. In this study, we thoroughly characterize the structural footprint of an anti-idiotype antibody (E1) specific for a potent, fully human DENV serotype 1-specific antibody, termed HM14c10, derived from a recovered patient. The crystal structure at a resolution of 2.5 Å of a complex between the Fab fragments of E1 and HM14c10 provides the first detailed molecular comparison of an anti-idiotype paratope specific for a human antibody with its analogous epitope, a discontinuous quaternary structure located at the surface of the viral particle that spans adjacent envelope (E) proteins. This comparison reveals that the footprints left by E1 and E on HM14c10 largely overlap, explaining why the formation of binary complexes is mutually exclusive. Structural mimicry of the DENV E epitope by the E1 combining site is achieved via the formation of numerous interactions with heavy chain complementarity domain regions (CDRs) of HM14c10, while fewer interactions are observed with its light chain than for the E protein. We show that E1 can be utilized to detect HM14c10-like antibodies in sera from patients who recovered from DENV-1, infection suggesting that this is a public (common) idiotype. These data demonstrate the utility of employing an anti-idiotype antibody to monitor a patient's specific immune responses and suggest routes for the improvement of E "mimicry" by E1 by increasing its recognition of the Fab HM14c10 light chain CDRs.IMPORTANCE A chimeric yellow fever-dengue live-attenuated tetravalent vaccine is now being marketed. Dengue remains a significant public health problem, because protection conferred by this vaccine against the four circulating serotypes is uneven. Reliable tools must be developed to measure the immune responses of individuals exposed to DENV either via viral infection or through vaccination. Anti-idiotypic antibodies provide precision tools for analyzing the pharmacokinetics of antibodies in an immune response and also for measuring the amount of circulating anti-infective therapeutic antibodies. Here, we characterize how an anti-idiotypic antibody (E1) binds antibody HM14c10, which potently neutralizes DENV serotype 1. We report the crystal structure at a resolution of 2.5 Å of a complex between the Fab fragments of E1 and HM14c10 and provide the first detailed molecular comparison between the anti-idiotype surface and its analogous epitope located at the surface of the dengue virus particle.

Contributions of Charged Residues in Structurally Dynamic Capsid Surface Loops to Rous Sarcoma Virus Assembly.

UNLABELLED: Extensive studies of orthoretroviral capsids have shown that many regions of the CA protein play unique roles at different points in the virus life cycle. The N-terminal domain (NTD) flexible-loop (FL) region is one such example: exposed on the outer capsid surface, it has been implicated in Gag-mediated particle assembly, capsid maturation, and early replication events. We have now defined the contributions of charged residues in the FL region of the Rous sarcoma virus (RSV) CA to particle assembly. Effects of mutations on assembly were assessed in vivo and in vitro and analyzed in light of new RSV Gag lattice models. Virus replication was strongly dependent on the preservation of charge at a few critical positions in Gag-Gag interfaces. In particular, a cluster of charges at the beginning of FL contributes to an extensive electrostatic network that is important for robust Gag assembly and subsequent capsid maturation. Second-site suppressor analysis suggests that one of these charged residues, D87, has distal influence on interhexamer interactions involving helix α7. Overall, the tolerance of FL to most mutations is consistent with current models of Gag lattice structures. However, the results support the interpretation that virus evolution has achieved a charge distribution across the capsid surface that (i) permits the packing of NTD domains in the outer layer of the Gag shell, (ii) directs the maturational rearrangements of the NTDs that yield a functional core structure, and (iii) supports capsid function during the early stages of virus infection. IMPORTANCE: The production of infectious retrovirus particles is a complex process, a choreography of protein and nucleic acid that occurs in two distinct stages: formation and release from the cell of an immature particle followed by an extracellular maturation phase during which the virion proteins and nucleic acids undergo major rearrangements that activate the infectious potential of the virion. This study examines the contributions of charged amino acids on the surface of the Rous sarcoma virus capsid protein in the assembly of appropriately formed immature particles and the maturational transitions that create a functional virion. The results provide important biological evidence in support of recent structural models of the RSV immature virions and further suggest that immature particle assembly and virion maturation are controlled by an extensive network of electrostatic interactions and long-range communication across the capsid surface.

Computational de novo design of antibodies binding to a peptide with high affinity.

Antibody drugs play a critical role in infectious diseases, cancer, autoimmune diseases, and inflammation. However, experimental methods for the generation of therapeutic antibodies such as using immunized mice or directed evolution remain time consuming and cannot target a specific antigen epitope. Here, we describe the application of a computational framework called OptMAVEn combined with molecular dynamics to de novo design antibodies. Our reference system is antibody 2D10, a single-chain antibody (scFv) that recognizes the dodecapeptide DVFYPYPYASGS, a peptide mimic of mannose-containing carbohydrates. Five de novo designed scFvs sharing less than 75% sequence similarity to all existing natural antibody sequences were generated using OptMAVEn and their binding to the dodecapeptide was experimentally characterized by biolayer interferometry and isothermal titration calorimetry. Among them, three scFvs show binding affinity to the dodecapeptide at the nM level. Critically, these de novo designed scFvs exhibit considerably diverse modeled binding modes with the dodecapeptide. The results demonstrate the potential of OptMAVEn for the de novo design of thermally and conformationally stable antibodies with high binding affinity to antigens and encourage the targeting of other antigen targets in the future. Biotechnol. Bioeng. 2017;114: 1331-1342. © 2017 Wiley Periodicals, Inc.

Elucidation of Lipid Binding Sites on Lung Surfactant Protein A Using X-ray Crystallography, Mutagenesis, and Molecular Dynamics Simulations.

Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen-associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air-liquid interface of the lung, ensures that the protein is poised for first-line interactions with inhaled pathogens. To improve our understanding of the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline headgroup through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions.

Atomic Detail Visualization of Photosynthetic Membranes with GPU-Accelerated Ray Tracing.

The cellular process responsible for providing energy for most life on Earth, namely photosynthetic light-harvesting, requires the cooperation of hundreds of proteins across an organelle, involving length and time scales spanning several orders of magnitude over quantum and classical regimes. Simulation and visualization of this fundamental energy conversion process pose many unique methodological and computational challenges. We present, in two accompanying movies, light-harvesting in the photosynthetic apparatus found in purple bacteria, the so-called chromatophore. The movies are the culmination of three decades of modeling efforts, featuring the collaboration of theoretical, experimental, and computational scientists. We describe the techniques that were used to build, simulate, analyze, and visualize the structures shown in the movies, and we highlight cases where scientific needs spurred the development of new parallel algorithms that efficiently harness GPU accelerators and petascale computers.

Molecular mechanisms of inhibition of influenza by surfactant protein D revealed by large-scale molecular dynamics simulation.

Surfactant protein D (SP-D), a mammalian C-type lectin, is the primary innate inhibitor of influenza A virus (IAV) in the lung. Interactions of SP-D with highly branched viral N-linked glycans on hemagglutinin (HA), an abundant IAV envelope protein and critical virulence factor, promote viral aggregation and neutralization through as yet unknown molecular mechanisms. Two truncated human SP-D forms, wild-type (WT) and double mutant D325A+R343V, representing neck and carbohydrate recognition domains are compared in this study. Whereas both WT and D325A+R343V bind to isolated glycosylated HA, WT does not inhibit IAV in neutralization assays; in contrast, D325A+R343V neutralization compares well with that of full-length native SP-D. To elucidate the mechanism for these biochemical observations, we have determined crystal structures of D325A+R343V in the presence and absence of a viral nonamannoside (Man9). On the basis of the D325A+R343V-Man9 structure and other crystallographic data, models of complexes between HA and WT or D325A+R343V were produced and subjected to molecular dynamics. Simulations reveal that whereas WT and D325A+R343V both block the sialic acid receptor site of HA, the D325A+R343V complex is more stable, with stronger binding caused by additional hydrogen bonds and hydrophobic interactions with HA residues. Furthermore, the blocking mechanism of HA differs for WT and D325A+R343V because of alternate glycan binding modes. The combined results suggest a mechanism through which the mode of SP-D-HA interaction could significantly influence viral aggregation and neutralization. These studies provide the first atomic-level molecular view of an innate host defense lectin inhibiting its viral glycoprotein target.

Rifaximin potentiates clarithromycin against Mycobacterium abscessus in vitro and in zebrafish.

Background: Mycobacterium abscessus is a non-tuberculous mycobacterium (NTM) that causes chronic pulmonary infections. Because of its extensive innate resistance to numerous antibiotics, treatment options are limited, often resulting in poor clinical outcomes. Current treatment regimens usually involve a combination of antibiotics, with clarithromycin being the cornerstone of NTM treatments. Objectives: To identify drug candidates that exhibit synergistic activity with clarithromycin against M. abscessus. Methods: We performed cell-based phenotypic screening of a compound library against M. abscessus induced to become resistant to clarithromycin. Furthermore, we evaluated the toxicity and efficacy of the top compound in a zebrafish embryo infection model. Results: The screen revealed rifaximin as a clarithromycin potentiator. The combination of rifaximin and clarithromycin was synergistic and bactericidal in vitro and potent in the zebrafish model. Conclusions: The data indicate that the rifaximin/clarithromycin combination is promising to effectively treat pulmonary NTM infections.

The mechanism of antiparallel ß-sheet formation based on conditioned self-avoiding walk.

By introducing an additional hydrogen bond to hydrogen bond interaction in the force field of the CSAW (Conditioned Self-Avoiding Walk) model, we investigate into the mechanism of antiparallel ß-sheet formation based on the folding of a short polyalanine in gas phase. Through our numerical simulation, we detect the possible presence of a transient helix during ß-sheet formation, whose presence is shown to have slowed the formation of ß-sheets by an order of magnitude. While we observe the mechanisms of nucleation, zipping and induction that drives the formation of a ß-sheet, we uncover a new mechanism that involves transient ß-turns and short ß-sheets during the formation of long ß-sheets. Our results have enabled us to provide an overview on the mechanisms of ß-sheet formation via two main folding pathways: slow folding through the intermediate state of transient helix, and fast folding from the nucleation of ß-turn.

Novel Phage Lysin Abp013 against Acinetobacter baumannii.

As antimicrobial resistance (AMR) continues to pose an ever-growing global health threat, propelling us into a post-antibiotic era, novel alternative therapeutic agents are urgently required. Lysins are bacteriophage-encoded peptidoglycan hydrolases that display great potential as a novel class of antimicrobials for therapeutics. While lysins against Gram-positive bacteria are highly effective when applied exogenously, it is challenging for lysins to access and cleave the peptidoglycan of Gram-negative bacteria due to their outer membrane. In this study, we identify a novel phage lysin Abp013 against Acinetobacter baumannii. Abp013 exhibited significant lytic activity against multidrug-resistant strains of A. baumannii. Notably, we found that Abp013 was able to tolerate the presence of human serum by up to 10%. Using confocal microscopy and LIVE/DEAD staining, we show that Abp013 can access and kill the bacterial cells residing in the biofilm. These results highlight the intrinsic bacteriolytic property of Abp013, suggesting the promising use of Abp013 as a novel therapeutic agent.

Engineering SARS-CoV-2 cocktail antibodies into a bispecific format improves neutralizing potency and breadth.

One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce antibody resistance. We engineered two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD). However, bsAb with the IgG-(scFv) 2 design (14-H-06) but not the CrossMAb design (14-crs-06) increases antigen-binding and virus-neutralizing activities and spectrum against multiple SARS-CoV-2 variants including the Omicron, than the cocktail. X-ray crystallography and computational simulations reveal distinct neutralizing mechanisms for individual cocktail antibodies and suggest higher inter-spike crosslinking potentials by 14-H-06 than 14-crs-06. In mouse models of infections by SARS-CoV-2 and the Beta, Gamma, and Delta variants, 14-H-06 exhibits higher or equivalent therapeutic efficacy than the cocktail. Rationally engineered bsAbs represent a cost-effective alternative to antibody cocktails and a promising strategy to improve potency and breadth.

Engineering SARS-CoV-2 specific cocktail antibodies into a bispecific format improves neutralizing potency and breadth.

One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce emergence of antibody resistance. Here we engineer two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD). However, bsAb with the IgG-(scFv)2 design (14-H-06) but not the CrossMAb design (14-crs-06) shows increased antigen-binding and virus-neutralizing activities against multiple SARS-CoV-2 variants as well as increased breadth of neutralizing activity compared to the cocktail. X-ray crystallography and cryo-EM reveal distinct binding models for individual cocktail antibodies, and computational simulations suggest higher inter-spike crosslinking potentials by 14-H-06 than 14-crs-06. In mouse models of infections by SARS-CoV-2 and multiple variants, 14-H-06 exhibits higher or equivalent therapeutic efficacy than the cocktail. Rationally engineered bsAbs represent a cost-effective alternative to antibody cocktails and a promising strategy to improve potency and breadth.

inPhocus: Current State and Challenges of Phage Research in Singapore.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion.

All-Atom MD Simulations of the HBV Capsid Complexed with AT130 Reveal Secondary and Tertiary Structural Changes and Mechanisms of Allostery.

The hepatitis B virus (HBV) capsid is an attractive drug target, relevant to combating viral hepatitis as a major public health concern. Among small molecules known to interfere with capsid assembly, the phenylpropenamides, including AT130, represent an important antiviral paradigm based on disrupting the timing of genome packaging. Here, all-atom molecular dynamics simulations of an intact AT130-bound HBV capsid reveal that the compound increases spike flexibility and improves recovery of helical secondary structure in the spike tips. Regions of the capsid-incorporated dimer that undergo correlated motion correspond to established sub-domains that pivot around the central chassis. AT130 alters patterns of correlated motion and other essential dynamics. A new conformational state of the dimer is identified, which can lead to dramatic opening of the intradimer interface and disruption of communication within the spike tip. A novel salt bridge is also discovered, which can mediate contact between the spike tip and fulcrum even in closed conformations, revealing a mechanism of direct communication across these sub-domains. Altogether, results describe a dynamical connection between the intra- and interdimer interfaces and enable mapping of allostery traversing the entire core protein dimer.

Engineered Lysins With Customized Lytic Activities Against Enterococci and Staphylococci.

The emergence of multidrug-resistant bacteria has made minor bacterial infections incurable with many existing antibiotics. Lysins are phage-encoded peptidoglycan hydrolases that have demonstrated therapeutic potential as a novel class of antimicrobials. The modular architecture of lysins enables the functional domains - catalytic domain (CD) and cell wall binding domain (CBD) - to be shuffled to create novel lysins. The CD is classically thought to be only involved in peptidoglycan hydrolysis whereas the CBD dictates the lytic spectrum of a lysin. While there are many studies that extended the lytic spectrum of a lysin by domain swapping, few have managed to introduce species specificity in a chimeric lysin. In this work, we constructed two chimeric lysins by swapping the CBDs of two parent lysins with different lytic spectra against enterococci and staphylococci. We showed that these chimeric lysins exhibited customized lytic spectra distinct from the parent lysins. Notably, the chimeric lysin P10N-V12C, which comprises a narrow-spectrum CD fused with a broad-spectrum CBD, displayed species specificity not lysing Enterococcus faecium while targeting Enterococcus faecalis and staphylococci. Such species specificity can be attributed to the narrow-spectrum CD of the chimeric lysin. Using flow cytometry and confocal microscopy, we found that the E. faecium cells that were treated with P10N-V12C are less viable with compromised membranes yet remained morphologically intact. Our results suggest that while the CBD is a major determinant of the lytic spectrum of a lysin, the CD is also responsible in the composition of the final lytic spectrum, especially when it pertains to species-specificity.

The SiaABC threonine phosphorylation pathway controls biofilm formation in response to carbon availability in Pseudomonas aeruginosa.

The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen.