Justin Lecher

The Crystal Structure of UehA in Complex with Ectoine—A Comparison with Other TRAP-T Binding Proteins

Substrate-binding proteins or extracellular solute receptors (ESRs) are components of both ABC (ATP binding cassette) and TRAP-T (tripartite ATP-independent periplasmic transporter). The TRAP-T system UehABC from Silicibacter pomeroyi DSS-3 imports the compatible solutes ectoine and 5-hydroxyectoine as nutrients. UehA, the ESR of the UehABC operon, binds both ectoine and 5-hydroxyectoine with high affinity (K(d) values of 1.4+/-0.1 and 1.1+/-0.1 microM, respectively) and delivers them to the TRAP-T complex. The crystal structure of UehA in complex with ectoine was determined at 2.9-A resolution and revealed an overall fold common for all ESR proteins from TRAP systems determined so far. A comparison of the recently described structure of TeaA from Halomonas elongata and an ectoine-binding protein (EhuB) from an ABC transporter revealed a conserved ligand binding mode that involves both directed and cation-pi interactions. Furthermore, a comparison with other known TRAP-T ESRs revealed a helix that might act as a selectivity filter imposing restraints on the ESRs that fine-tune ligand recognition and binding and finally might determine the selection of the cognate substrate.

The Compatible-Solute-Binding Protein OpuAC from Bacillus subtilis: Ligand Binding, Site-Directed Mutagenesis, and Crystallographic Studies

In the soil bacterium Bacillus subtilis, five transport systems work in concert to mediate the import of various compatible solutes that counteract the deleterious effects of increases in the osmolarity of the environment. Among these five systems, the ABC transporter OpuA, which catalyzes the import of glycine betaine and proline betaine, has been studied in detail in the past. Here, we demonstrate that OpuA is capable of importing the sulfobetaine dimethylsulfonioacetate (DMSA). Since OpuA is a classic ABC importer that relies on a substrate-binding protein priming the transporter with specificity and selectivity, we analyzed the OpuA-binding protein OpuAC by structural and mutational means with respect to DMSA binding. The determined crystal structure of OpuAC in complex with DMSA at a 2.8-A resolution and a detailed mutational analysis of these residues revealed a hierarchy within the amino acids participating in substrate binding. This finding is different from those for other binding proteins that recognize compatible solutes. Furthermore, important principles that enable OpuAC to specifically bind various compatible solutes were uncovered.

Sequestration of a β-hairpin for control of α-synuclein aggregation.

The misfolding and aggregation of the protein α-synuclein (α-syn), which results in the formation of amyloid fibrils, is involved in the pathogenesis of Parkinsons disease and other synucleinopathies. The emergence of amyloid toxicity is associated with the formation of partially folded aggregation intermediates. Here, we engineered a class of binding proteins termed β-wrapins (β-wrap proteins) with affinity for α-synuclein (α-syn). The NMR structure of an α-syn:β-wrapin complex reveals a β-hairpin of α-syn comprising the sequence region α-syn(37-54). The β-wrapin inhibits α-syn aggregation and toxicity at substoichiometric concentrations, demonstrating that it interferes with the nucleation of aggregation.

Structural insights into conformational changes of a cyclic nucleotide-binding domain in solution from Mesorhizobium loti K1 channel

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are activated by binding of ligands to a domain (CNBD) located on the cytoplasmic side of the channel. The underlying mechanisms are not well understood. To elucidate the gating mechanism, structures of both the ligand-free and -bound CNBD are required. Several crystal structures of the CNBD from HCN2 and a bacterial CNG channel (MloK1) have been solved. However, for HCN2, the cAMP-free and -bound state did not reveal substantial structural rearrangements. For MloK1, structural information for the cAMP-free state has only been gained from mutant CNBDs. Moreover, in the crystal, the CNBD molecules form an interface between dimers, proposed to be important for allosteric channel gating. Here, we have determined the solution structure by NMR spectroscopy of the cAMP-free wild-type CNBD of MloK1. A comparison of the solution structure of cAMP-free and -bound states reveals large conformational rearrangement on ligand binding. The two structures provide insights on a unique set of conformational events that accompany gating within the ligand-binding site.

The Nedd4-1 WW Domain Recognizes the PY Motif Peptide through Coupled Folding and Binding Equilibria.

The four WW domains of human Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) interact with the PPxY (PY) motifs of the human epithelial Na(+) channel (hENaC) subunits, with the third WW domain (WW3*) showing the highest affinity. We have shown previously that the α-hENaC PY motif binding interface of WW3* undergoes conformational exchange on the millisecond time scale, indicating that conformational sampling plays a role in peptide recognition. To further understand this role, the structure and dynamics of hNedd4-1 WW3* were investigated. The nuclear Overhauser effect-derived structure of apo-WW3* resembles the domain in complex with the α-hENaC peptide, although particular side chain conformations change upon peptide binding, which was further investigated by molecular dynamics simulations. Model-free analysis of the (15)N nuclear magnetic resonance spin relaxation data showed that the apo and peptide-bound states of WW3* have similar backbone picosecond to nanosecond time scale dynamics. However, apo-WW3* exhibits pronounced chemical exchange on the millisecond time scale that is quenched upon peptide binding. (1)HN and (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments at various temperatures revealed that apo-WW3* exists in an equilibrium between the natively folded peptide binding-competent state and a random coil-like denatured state. The thermodynamics of the folding equilibrium was determined by fitting a thermal denaturation profile monitored by circular dichroism spectroscopy in combination with the CPMG data, leading to the conclusion that the unfolded state is populated to ∼ 20% at 37 °C. These results show that the binding of the hNedd4-1 WW3* domain to α-hENaC is coupled to the folding equilibrium.

Pyroglutamate-Modified Amyloid-β(3–42) Shows α-Helical Intermediates before Amyloid Formation

Pyroglutamate-modified amyloid-β (pEAβ) has been described as a relevant Aβ species in Alzheimers-disease-affected brains, with pEAβ (3-42) as a dominant isoform. Aβ (1-40) and Aβ (1-42) have been well characterized under various solution conditions, including aqueous solutions containing trifluoroethanol (TFE). To characterize structural properties of pEAβ (3-42) possibly underlying its drastically increased aggregation propensity compared to Aβ (1-42), we started our studies in various TFE-water mixtures and found striking differences between the two Aβ species. Soluble pEAβ (3-42) has an increased tendency to form β-sheet-rich structures compared to Aβ (1-42), as indicated by circular dichroism spectroscopy data. Kinetic assays monitored by thioflavin-T show drastically accelerated aggregation leading to large fibrils visualized by electron microscopy of pEAβ (3-42) in contrast to Aβ (1-42). NMR spectroscopy was performed for backbone and side-chain chemical-shift assignments of monomeric pEAβ (3-42) in 40% TFE solution. Although the difference between pEAβ (3-42) and Aβ (1-42) is purely N-terminal, it has a significant impact on the chemical environment of >20% of the total amino acid residues, as revealed by their NMR chemical-shift differences. Freshly dissolved pEAβ (3-42) contains two α-helical regions connected by a flexible linker, whereas the N-terminus remains unstructured. We found that these α-helices act as a transient intermediate to β-sheet and fibril formation of pEAβ (3-42).

Conformational Polymorphism in Autophagy-Related Protein GATE-16

Autophagy is a fundamental homeostatic process in eukaryotic organisms, fulfilling essential roles in development and adaptation to stress. Among other factors, formation of autophagosomes critically depends on proteins of the Atg8 (autophagy-related protein 8) family, which are reversibly conjugated to membrane lipids. We have applied X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to study the conformational dynamics of Atg8-type proteins, using GATE-16 (Golgi-associated ATPase enhancer of 16 kDa), also known as GABARAPL2, as a model system. This combination of complementary approaches provides new insight into a structural transition centered on the C-terminus, which is crucial for the biological activity of these proteins.

Resonance assignments of the nucleotide-free wildtype MloK1 cyclic nucleotide-binding domain.

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels play crucial roles in neuronal excitability and signal transduction of sensory cells. These channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). A comparison of the structures of wildtype ligand-free and ligand-bound CNBD is essential to elucidate the mechanism underlying nucleotide-dependent activation of CNBDs. We recently reported the solution structure of the Mesorhizobium loti K1 (MloK1) channel CNBD in complex with cAMP. We have now extended these studies and achieved nearly complete assignments of 1H, 13C and 15N resonances of the nucleotide-free CNBD. A completely new assignment of the nucleotide-free wildtype CNBD was necessary due to the sizable chemical shift differences as compared to the cAMP bound CNBD and the slow exchange behaviour between both forms. Scattering of these chemical shift differences over the complete CNBD suggests that nucleotide binding induces significant overall conformational changes.

1H, 15N and 13C resonance assignment of the N-terminal C39 peptidase-like domain of the ABC transporter Haemolysin B (HlyB).

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins, which catalyze the translocation of molecules across biological membranes in an ATP-dependent manner. Despite the diversity in the transported substrates, they all share the same architecture, comprised of two transmembrane (TMD) and two nucleotide-binding domains (NBD). Members of the bacteriocin ABC transporter subfamily feature a special domain, belonging to the C39 (cystein protease family 39) peptidase protein family. These domains are assumed to cleave a C-terminal signal sequence from the protein or peptide substrate before or during the transport process. Although the C39 peptidase-like domain of the ABC transporter haemolysin B from E. coli shows no proteolytic activity, it is essential for the function of this transporter. In order to elucidate the contribution of the isolated C39 peptidase-like domain in the whole transport process, the backbone and side chain 1H, 15N and 13C-NMR chemical shifts have been assigned.

Data describing the solution structure of the WW3* domain from human Nedd4-1.

The third WW domain (WW3*) of human Nedd4-1 (Neuronal precursor cell expressed developmentally down-regulated gene 4-1) interacts with the poly-proline (PY) motifs of the human epithelial Na+ channel (hENaC) subunits at micromolar affinity. This data supplements the article (Panwalkar et al., 2015) [1]. We describe the NMR experiments used to solve the solution structure of the WW3* domain. We also present NOE network data for defining the rotameric state of side chains of peptide binding residues, and complement this data with χ1 dihedral angles derived from 3J couplings and molecular dynamics simulations data.