Transmembrane helix dynamics of bacterial chemoreceptors supports a piston model of signalling.

Transmembrane α-helices play a key role in many receptors, transmitting a signal from one side to the other of the lipid bilayer membrane. Bacterial chemoreceptors are one of the best studied such systems, with a wealth of biophysical and mutational data indicating a key role for the TM2 helix in signalling. In particular, aromatic (Trp and Tyr) and basic (Arg) residues help to lock α-helices into a membrane. Mutants in TM2 of E. coli Tar and related chemoreceptors involving these residues implicate changes in helix location and/or orientation in signalling. We have investigated the detailed structural basis of this via high throughput coarse-grained molecular dynamics (CG-MD) of Tar TM2 and its mutants in lipid bilayers. We focus on the position (shift) and orientation (tilt, rotation) of TM2 relative to the bilayer and how these are perturbed in mutants relative to the wildtype. The simulations reveal a clear correlation between small (ca. 1.5 Å) shift in position of TM2 along the bilayer normal and downstream changes in signalling activity. Weaker correlations are seen with helix tilt, and little/none between signalling and helix twist. This analysis of relatively subtle changes was only possible because the high throughput simulation method allowed us to run large (n = 100) ensembles for substantial numbers of different helix sequences, amounting to ca. 2000 simulations in total. Overall, this analysis supports a swinging-piston model of transmembrane signalling by Tar and related chemoreceptors

The role of protein-membrane interactions in modulation of signaling by bacterial chemoreceptors

Environmental signals are sensed by membrane-spanning receptors that communicate with the cell interior. Bacterial chemoreceptors modulate the activity of the CheA kinase in response to binding of small ligands or upon interaction with substrate-bound periplasmic-binding proteins. The mechanism of signal transduction across the membrane is a displacement of the second transmembrane domain (TM2) a few angstroms toward the cytoplasm. This movement repositions a dynamic transmembrane helix relative to the plane of the cell membrane. The research presented in this dissertation investigated the contribution of TM2-membrane interactions to signaling by the aspartate chemoreceptor (Tar) of Escherichia coli. Aromatic residues that reside at the cytoplasmic polar-hydrophobic membrane interface (Trp-209 and Tyr-210) were found to play a significant role in regulating signaling by Tar. These interactions were subsequently manipulated to modulate the signaling properties of Tar. The baseline signaling state was shown to be incrementally altered by repositioning the Trp-209/Tyr-210 pair. To our knowledge, this is the first example of harnessing membrane-protein interactions to modulate the signal output of a transmembrane receptor in a controlled and predictable manner. Potential long-term applications include the use of analogous mutations to elucidate two-component signaling pathways, to engineer the signaling parameters of biosensors that incorporate chemoreceptors, and to predict the movement of dynamic transmembrane helices in silico

The Role of the TM2-HAMP Junction in Control of the Signaling State of the Aspartate Chemoreceptor of E. coli

The mechanism of allosteric coupling between the external ligand-binding domain and the internal signaling domain of bacterial chemoreceptors is poorly understood. Genetic, biochemical, and biophysical evidence suggests that transmembrane helix 2 (TM2) undergoes a piston-like displacement of approximately 1-3 Angstroms toward the cytoplasm upon the binding of aspartate to the Tar receptor. The signal is then transmitted to the cytoplasmic signaling domain via the HAMP domain, a conserved motif found in all methyl-accepting chemotaxis proteins (MCPs) and most histidine protein kinases (HPKs). HAMP forms a parallel four-helix bundle consisting of a dimer of two amphipathic helices (AS1 and AS2) connected by a flexible linker. The MLLT sequence between residues Arg-214, at the end of TM2, and the conserved residue Pro-219, at the beginning of AS1 of the HAMP domain (the TM2-HAMP junction), is predicted to be able to form a helical extension of TM2. We hypothesized that perturbing the native secondary structure and/or the length of the TM2-HAMP junction would disrupt the ability of HAMP to communicate the input signal from TM2 to the kinase-control domain. To test this hypothesis, we designed two experiments. First, constructs were made in which 1 to 3 Gly residues were inserted between T218 and P219. Second, Tar variants were constructed in which 1 to 9 Gly residues were inserted between R214 and P219. The results suggest that increasing the length and flexibility of the TM2-HAMP connection tends to uncouple signal propagation between the TM2 and the HAMP elements and suggests that HAMP alone causes an inhibitory effect on the cytoplasmic signaling domain. To determine whether the predicted helical register of the MLLT sequence is an important component of the propagation of the transmembrane signal from TM2 to the HAMP domain, we added and subtracted helical residues to the MLLT sequence. The results suggest that helical register and length of the TM2-HAMP junction are essential for optimal receptor function

Application of Computational Molecular Biophysics to Problems in Bacterial Chemotaxis

The combination of physics, biology, chemistry, and computer science constitutes the promising field of computational molecular biophysics. This field studies the molecular properties of DNA, protein lipids and biomolecules using computational methods. For this dissertation, I approached four problems involving the chemotaxis pathway, the set of proteins that function as the navigation system of bacteria and lower eukaryotes. In the first chapter, I used a special-purpose machine for molecular dynamics simulations, Anton, to simulate the signaling domain of the chemoreceptor in different signaling states for a total of 6 microseconds. Among other findings, this study provides enough evidence to propose a novel molecular mechanism for the kinase activation by the chemoreceptor and reconcile previously conflicting experimental data. In the second chapter, my molecular dynamics studies of the scaffold protein cheW reveals the existence and role of a conserved salt-bridge that stabilizes the relative position of the two binding sites in the chew surface: the chemoreceptor and the kinase. The results were further confirmed with NMR experiments performed with collaborators at the University of California in Santa Barbara, CA. In the third chapter, my colleagues and I investigate the quality of homology modeled structures with cheW protein as a benchmark. By subjecting the models to molecular dynamics and Monte Carlo simulations, we show that the homology models are snapshots of a larger ensemble of conformations very similar to the one generated by the experimental structures. In the fourth chapter, I use bioinformatics and basic mathematical modeling to predict the specific chemoreceptor(s) expressed in vivo and imaged with electron cryo tomography (ECT) by our collaborators at the California Institute of Technology. The study was essential to validate the argument that the hexagonal arrangement of transmembrane chemoreceptors is universal among bacteria, a major breakthrough in the field of chemotaxis. In summary, this thesis presents a collection of four works in the field of bacterial chemotaxis where either methods of physics or the quantitative approach of physicists were of fundamental importance for the success of the project

The C-Terminus of Transmembrane Helix 2 (TM2) of the Escherichia coli Tar Chemorecptor Determines Signal Output and Ligand Sensitivity

Methyl-accepting chemotaxis proteins MCPs can bind one or more receptor- specific ligands. In the case of the Tar MCP of Escherichia coli (TarEc), a primary attractant ligand is aspartate. Its binding to the periplasmic domain of Tar generates a conformational change that is transmitted via helix 4 transmembrane helix 2 (TM2). An inward movement of TM2 initiates a transmembrane signal to the cytoplasmic HAMP (histidine kinases, adenyl cyclases, methyl-accepting proteins, phosphatases) domain. Baseline CheA kinase-stimulating activity and ligand-induced responses are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). The cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in TarEc, is of particular importance. These residues are not highly conserved among transmembrane receptors having a HAMP domain, although there are almost always some aromatic residues in this region. The question thus becomes what properties of this aromatic anchor are necessary for proper signal transduction. In this dissertation, I studied the effect on TarEc function by substituting all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. This library of TarEc variants allowed the direct assessment of the effect of the residue composition of the aromatic anchor and led to a model of how the wild-type anchor maintains the base-line signaling state in TarEc. Additional receptor variants containing double aromatic tandems and Ala substitutions for the periplasmic Trp residue were created, and the aromatic residues were also shifted in position within the six residues 207-212. Trp, Tyr, and Phe, in that order, had the greatest effect on function when they were moved to novel positions. It was also discovered that Gly-211 plays a critical role in maintaining receptor function. A model was generated that proposes that Gly-211 plays a role in maintaining the flexibility of the TM2-HAMP domain connector. The results suggest that the signaling properties of the transmembrane sensor kinases of two-component systems can be predicted by the nature of their TM2-HAMP connections. It may also be possible to modulate their activity in a controlled way by manipulating the amino acid sequences that comprise those connections

CHEMORECEPTOR DYNAMICS AND SIGNALING: NMR MEASUREMENTS WITHIN FUNCTIONAL COMPLEXES

Bacteria employ remarkable membrane-bound nanoarrays to sense their environment and direct their swimming. Arrays consist of chemotaxis receptor trimers of dimers that are bridged at their membrane-distal tips by rings of two cytoplasmic proteins, a kinase CheA and a coupling protein CheW. It is widely accepted that ligand binding to the receptor causes a 2 Å piston motion of a helix that extends through the periplasmic and transmembrane domains. However, it is not clear how the signal propagates 200 Å further to control activity of the kinase bound at the tip of the receptor. Dynamic changes within the cytoplasmic domain of the receptor have been proposed to play a key role in signal transmission. To test these proposals, we applied solid-state NMR to study the structure and dynamics of the E coli Asp receptor cytoplasmic fragment (U-13C,15N-CF) in native-like arrays of functional complexes with CheA and CheW. To detect segments that experience motions on the millisecond time scale, we use a 15N{13C} REDOR filter to remove signals from rigid backbone carbons and retain signals from backbone carbons with ms-timescale dynamics. This experiment exhibits only 60-70% of the expected REDOR dephasing, suggesting that 40-30% of the backbone has ms-timescale mobility that averages the ~ 1000 Hz 13C15N dipolar couplings. A novel REDOR-filtered DARR experiment led us to identify MH2 (methylation helix 2) as the region with ms-timescale motion. INEPT spectra reveal dynamics on the ns or shorter timescale. The INEPT-detectable regions are identified through a combination of biochemical and NMR approaches, to establish that MH1 (methylation helix 1) is mobile on the ns timescale. Interestingly, the INEPT and REDOR studies indicate the methylation region has asymmetric mobility: MH1 has ns-timescale dynamics that increase in the kinase-off state, and MH2 has ms-timescale dynamics that decrease in the kinase-off state. Thus these NMR measurements of the dynamics of CF within its functional complexes provide insight into the structural organization and signaling-related changes of the receptor methylation region, which is responsible both for transmitting the ligand-binding signal and for adaptation, both of which are critical to the chemotaxis response

The Role of the TM2-HAMP Junction in Control of the Signaling State of the Aspartate Chemoreceptor of E. coli

The mechanism of allosteric coupling between the external ligand-binding domain and the internal signaling domain of bacterial chemoreceptors is poorly understood. Genetic, biochemical, and biophysical evidence suggests that transmembrane helix 2 (TM2) undergoes a piston-like displacement of approximately 1-3 Angstroms toward the cytoplasm upon the binding of aspartate to the Tar receptor. The signal is then transmitted to the cytoplasmic signaling domain via the HAMP domain, a conserved motif found in all methyl-accepting chemotaxis proteins (MCPs) and most histidine protein kinases (HPKs). HAMP forms a parallel four-helix bundle consisting of a dimer of two amphipathic helices (AS1 and AS2) connected by a flexible linker. The MLLT sequence between residues Arg-214, at the end of TM2, and the conserved residue Pro-219, at the beginning of AS1 of the HAMP domain (the TM2-HAMP junction), is predicted to be able to form a helical extension of TM2. We hypothesized that perturbing the native secondary structure and/or the length of the TM2-HAMP junction would disrupt the ability of HAMP to communicate the input signal from TM2 to the kinase-control domain. To test this hypothesis, we designed two experiments. First, constructs were made in which 1 to 3 Gly residues were inserted between T218 and P219. Second, Tar variants were constructed in which 1 to 9 Gly residues were inserted between R214 and P219. The results suggest that increasing the length and flexibility of the TM2-HAMP connection tends to uncouple signal propagation between the TM2 and the HAMP elements and suggests that HAMP alone causes an inhibitory effect on the cytoplasmic signaling domain. To determine whether the predicted helical register of the MLLT sequence is an important component of the propagation of the transmembrane signal from TM2 to the HAMP domain, we added and subtracted helical residues to the MLLT sequence. The results suggest that helical register and length of the TM2-HAMP junction are essential for optimal receptor function

Elucidating the Energetics of Bacterial Signal Transduction: Insights From Phoq

Bacteria transduce signals across the membrane using two-component systems, consisting of a membrane-spanning sensor histidine kinase and a cytoplasmic response regulator. The histidine kinase, PhoQ, serves as a master regulator of virulence response in S. typhimurium and E. coli. It also is inhibited by divalent cations, particularly Mg2+. While the periplasmic sensor domain of this protein has a unique function, the cytoplasmic portion of this modular protein is made of structurally conserved domains found in many other bacterial sensor kinases. Signal transduction through these conserved domains is thought to be universal; however, the structural and energetic rearrangements that occur during signaling have generated numerous models. Through Bayesian inference we constructed a two-state model based on cysteine crosslinking data and homologous crystal structures. These two signaling states differ in membrane depth of the periplasmic acidic patch as well as the reciprocal displacement of diagonal helices along the dimer interface. Comparative studies of multiple histidine kinases suggest that diagonal displacement of helices is a common mode of signal transduction. A similar scissor-like model was previously ruled out in CheA-linked chemoreceptors; therefore, this new evidence suggests that sensor His-kinase and CheA-linked receptors possess different signaling mechanisms. To unify the various signaling mechanisms that exist for the different protein domains, we built a thermodynamic model based on Linked Equilibrating Domains (LED). We used this model to quantitatively interpret functional data of single-point Ala, Phe and Cys mutants throughout the signal transducing regions of PhoQ. Data from 35 mutants, including both activating and deactivating phenotypes, were globally fit using LED, and gross features such as Vmax and Kd were related to more nuanced population distributions and thermodynamic coupling. LED analysis highlights the principles by which individual signaling domains can be connected to create a functional signal transducer. These principles allow us to quantitatively explain signaling in histidine kinases and are likely to be broadly applicable to many other signal transduction proteins