Kylie J. Watts

Structure of Concatenated HAMP Domains Provides a Mechanism for Signal Transduction

HAMP domains are widespread prokaryotic signaling modules found as single domains or poly-HAMP chains in both transmembrane and soluble proteins. The crystal structure of a three-unit poly-HAMP chain from the Pseudomonas aeruginosa soluble receptor Aer2 defines a universal parallel four-helix bundle architecture for diverse HAMP domains. Two contiguous domains integrate to form a concatenated di-HAMP structure. The three HAMP domains display two distinct conformations that differ by changes in helical register, crossing angle, and rotation. These conformations are stabilized by different subsets of conserved residues. Known signals delivered to HAMP would be expected to switch the relative stability of the two conformations and the position of a coiled-coil phase stutter at the junction with downstream helices. We propose that the two conformations represent opposing HAMP signaling states and suggest a signaling mechanism whereby HAMP domains interconvert between the two states, which alternate down a poly-HAMP chain.

Structure-Function Relationships in the HAMP and Proximal Signaling Domains of the Aerotaxis Receptor Aer

Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two alpha-helices separated by a structured loop. The proximal signaling domain consisted of two alpha-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the beta-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed.

HAMP Domain Conformers That Propagate Opposite Signals in Bacterial Chemoreceptors

How do cell-surface receptors transmit signals into cells? This study resolves how signal relay occurs through the HAMP domains of bacterial chemoreceptors by causing them to switch between two conformational states.

Interactions between the PAS and HAMP domains of the Escherichia coli aerotaxis receptor Aer.

The Escherichia coli energy-sensing Aer protein initiates aerotaxis towards environments supporting optimal cellular energy. The Aer sensor is an N-terminal, FAD-binding, PAS domain. The PAS domain is linked by an F1 region to a membrane anchor, and in the C-terminal half of Aer, a HAMP domain links the membrane anchor to the signaling domain. The F1 region, membrane anchor, and HAMP domain are required for FAD binding. Presumably, alterations in the redox potential of FAD induce conformational changes in the PAS domain that are transmitted to the HAMP and C-terminal signaling domains. In this study we used random mutagenesis and intragenic pseudoreversion analysis to examine functional interactions between the HAMP domain and the N-terminal half of Aer. Missense mutations in the HAMP domain clustered in the AS-2 alpha-helix and abolished FAD binding to Aer, as previously reported. Three amino acid replacements in the Aer-PAS domain, S28G, A65V, and A99V, restored FAD binding and aerotaxis to the HAMP mutants. These suppressors are predicted to surround a cleft in the PAS domain that may bind FAD. On the other hand, suppression of an Aer-C253R HAMP mutant was specific to an N34D substitution with a predicted location on the PAS surface, suggesting that residues C253 and N34 interact or are in close proximity. No suppressor mutations were identified in the F1 region or membrane anchor. We propose that functional interactions between the PAS domain and the HAMP AS-2 helix are required for FAD binding and aerotactic signaling by Aer.

Sequence variation and physical state of human papillomavirus type 16 cervical cancer isolates from Australia and New Caledonia

Sequence diversity over 2600 nucleotides of the upstream regulatory region (URR) and the E6 and E2/E4 genes of 34 human papillomavirus (HPV)16 cervical cancer isolates from Australia and New Caledonia was investigated. One 81 base duplication, 41 single base substitutions and 1 single base insertion were identified in the URRs. Some of these changes are reported here for the first time. Several of the 19 changes impacting transcription factor binding sites had the potential to alter promoter activity. Twenty‐eight (82%) of the variants belonged to the European lineage, 4 (12%) were Asian and 2 (6%) were Asian‐American. Eighteen of 27 (67%) isolates where the E6 gene was examined contained amino acid substitutions. Of 13 isolates sequenced with intact E2 genes, 12 (92%) contained amino acid substitutions in the E2 protein and 3 (23%) amino acid substitutions in the overlapping E4 protein. Some of the changes in E6 and E2 may alter immunological epitopes or protein function. The physical state of HPV DNA was assessed by Southern hybridization and PCR for an intact E2 gene. Overall, 11 of 25 isolates contained only integrated HPV DNA, 10 only episomal HPV DNA and 4 both integrated and episomal DNA. No particular patterns of variation in the URR, E6 or E2/E4 genes predicted physical state. This investigation represents one of the most comprehensive studies of its kind and fills an important gap in global sequence data.

PAS/poly-HAMP signalling in Aer-2, a soluble haem-based sensor

Poly‐HAMP domains are widespread in bacterial chemoreceptors, but previous studies have focused on receptors with single HAMP domains. The Pseudomonas aeruginosa chemoreceptor, Aer‐2, has an unusual domain architecture consisting of a PAS‐sensing domain sandwiched between three N‐terminal and two C‐terminal HAMP domains, followed by a conserved kinase control module. The structure of the N‐terminal HAMP domains was recently solved, making Aer‐2 the first protein with resolved poly‐HAMP structure. The role of Aer‐2 in P. aeruginosa is unclear, but here we show that Aer‐2 can interact with the chemotaxis system of Escherichia coli to mediate repellent responses to oxygen, carbon monoxide and nitric oxide. Using this model system to investigate signalling and poly‐HAMP function, we determined that the Aer‐2 PAS domain binds penta‐co‐ordinated b‐type haem and that reversible signalling requires four of the five HAMP domains. Deleting HAMP 2 and/or 3 resulted in a kinase‐off phenotype, whereas deleting HAMP 4 and/or 5 resulted in a kinase‐on phenotype. Overall, these data support a model in which ligand‐bound Aer‐2 PAS and HAMP 2 and 3 act together to relieve inhibition of the kinase control module by HAMP 4 and 5, resulting in the kinase‐on state of the Aer‐2 receptor.

Oxygen and Redox Sensing by Two‐Component Systems That Regulate Behavioral Responses: Behavioral Assays and Structural Studies of Aer Using In Vivo Disulfide Cross‐Linking

A remarkable increase in the number of annotated aerotaxis (oxygen-seeking) and redox taxis sensors can be attributed to recent advances in bacterial genomics. However, in silico predictions should be supported by behavioral assays and genetic analyses that confirm an aerotaxis or redox taxis function. This chapter presents a collection of procedures that have been highly successful in characterizing aerotaxis and redox taxis in Escherichia coli. The methods are described in enough detail to enable investigators of other species to adapt the procedures for their use. A gas flow cell is used to quantitate the temporal responses of bacteria to a step increase or decrease in oxygen partial pressure or redox potential. Bacterial behavior in spatial gradients is analyzed using optically flat capillaries and soft agar plates (succinate agar or tryptone agar). We describe two approaches to estimate the preferred partial pressure of oxygen that attracts a bacterial species; this concentration is important for understanding microbial ecology. At the molecular level, we describe procedures used to determine the structure and topology of Aer, a membrane receptor for aerotaxis. Cysteine-scanning mutagenesis and in vivo disulfide cross-linking procedures utilize the oxidant Cu(II)-(1,10-phenanthroline)(3) and bifunctional sulfhydryl-reactive probes. Finally, we describe methods used to determine the boundaries of transmembrane segments of receptors such as Aer. These include 5-iodoacetamidofluorescein, 4-acetamido-4-disulfonic acid, disodium salt (AMS), and methoxy polyethylene glycol maleimide, a 5-kDa molecular mass probe that alters the mobility of Aer on SDS-PAGE.

Function of the N-Terminal Cap of the PAS Domain in Signaling by the Aerotaxis Receptor Aer

Aer, the Escherichia coli receptor for behavioral responses to oxygen (aerotaxis), energy, and redox potential, contains a PAS sensory-input domain. Within the PAS superfamily, the N-terminal segment (N-cap) is poorly conserved and its role is not well understood. We investigated the role of the N-cap (residues 1 to 19) in the Aer PAS domain by missense and truncation mutagenesis. Aer-PAS N-cap truncations and an Aer-M21P substitution resulted in low cellular levels of the mutant proteins, suggesting that the N-terminal region was important for stabilizing the structure of the PAS domain. The junction of the N-cap and PAS core was critical for signaling in Aer. Mutations and truncations in the sequence encoding residues 15 to 21 introduced a range of phenotypes, including defects in FAD binding, constant tumbling motility, and an inverse response in which E. coli cells migrated away from oxygen concentrations to which they are normally attracted. The proximity of two N-cap regions in an Aer dimer was assessed in vivo by oxidatively cross-linking serial cysteine substitutions. Cross-linking of several cysteine replacements at 23 degrees C was attenuated at 10 degrees C, indicating contact was not at a stable dimer interface but required lateral mobility. We observed large multimers of Aer when we combined cross-linking of N-cap residues with a cysteine replacement that cross-links exclusively at the Aer dimer interface. This suggests that the PAS N-cap faces outwards in a dimer and that PAS-PAS contacts can occur between adjacent dimers.

Minimal requirements for oxygen sensing by the aerotaxis receptor Aer

The PAS and HAMP domain superfamilies are signal transduction modules found in all kingdoms of life. The Aer receptor, which contains both domains, initiates rapid behavioural responses to oxygen (aerotaxis) and other electron acceptors, guiding Escherichia coli to niches where it can generate optimal cellular energy. We used intragenic complementation to investigate the signal transduction pathway from the Aer PAS domain to the signalling domain. These studies showed that the HAMP domain of one monomer in the Aer dimer stabilized FAD binding to the PAS domain of the cognate monomer. In contrast, the signal transduction pathway was intra‐subunit, involving the PAS and signalling domains from the same monomer. The minimal requirements for signalling were investigated in heterodimers containing a full‐length and truncated monomer. Either the PAS or signalling domains could be deleted from the non‐signalling subunit of the heterodimer, but removing 16 residues from the C‐terminus of the signalling subunit abolished aerotaxis. Although both HAMP domains were required for aerotaxis, signalling was not disrupted by missense mutations in the HAMP domain from the signalling subunit. Possible models for Aer signal transduction are compared.

Architecture of the Soluble Receptor Aer2 Indicates an In-Line Mechanism for PAS and HAMP Domain Signaling

Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here, we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS (Per-Arnt-Sim) and poly-HAMP (histidine kinase-adenylyl cyclase-methyl-accepting chemotaxis protein-phosphatase) domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state, identifies conformational changes induced by ligand binding that are likely essential for the signaling mechanism. Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS but propagate to the Iβ strand in a manner predicted to alter PAS-PAS associations and the downstream HAMP junction within full-length Aer2. Small-angle X-ray scattering of PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initio small-angle X-ray scattering molecular envelopes, and pulsed dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the Escherichia coli Aer paradigm, where side-on PAS-HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains.