The role of leucine and isoleucine in tuning the hydropathy of class A GPCRs.

Leucine and Isoleucine are two amino acids that differ only by the positioning of one methyl group. This small difference can have important consequences in α-helices, as the ß-branching of Ile results in helix destabilization. We set out to investigate whether there are general trends for the occurrences of Leu and Ile residues in the structures and sequences of class A GPCRs (G protein-coupled receptors). GPCRs are integral membrane proteins in which α-helices span the plasma membrane seven times and which play a crucial role in signal transmission. We found that Leu side chains are generally more exposed at the protein surface than Ile side chains. We explored whether this difference might be attributed to different functions of the two amino acids and tested if Leu tunes the hydrophobicity of the transmembrane domain based on the Wimley-White whole-residue hydrophobicity scales. Leu content decreases the variation in hydropathy between receptors and correlates with the non-Leu receptor hydropathy. Both measures indicate that hydropathy is tuned by Leu. To test this idea further, we generated protein sequences with random amino acid compositions using a simple numerical model, in which hydropathy was tuned by adjusting the number of Leu residues. The model was able to replicate the observations made with class A GPCR sequences. We speculate that the hydropathy of transmembrane domains of class A GPCRs is tuned by Leu (and to some lesser degree by Lys and Val) to facilitate correct insertion into membranes and/or to stably anchor the receptors within membranes.

Unexpected dynamics in femtomolar complexes of binding proteins with peptides.

Ultra-tight binding is usually observed for proteins associating with rigidified molecules. Previously, we demonstrated that femtomolar binders derived from the Armadillo repeat proteins (ArmRPs) can be designed to interact very tightly with fully flexible peptides. Here we show for ArmRPs with four and seven sequence-identical internal repeats that the peptide-ArmRP complexes display conformational dynamics. These dynamics stem from transient breakages of individual protein-residue contacts that are unrelated to overall unbinding. The labile contacts involve electrostatic interactions. We speculate that these dynamics allow attaining very high binding affinities, since they reduce entropic losses. Importantly, only NMR techniques can pick up these local events by directly detecting conformational exchange processes without complications from changes in solvent entropy. Furthermore, we demonstrate that the interaction surface of the repeat protein regularizes upon peptide binding to become more compatible with the peptide geometry. These results provide novel design principles for ultra-tight binders.

Side-chain dynamics of the α1B -adrenergic receptor determined by NMR via methyl relaxation.

G protein-coupled receptors (GPCRs) are medically important membrane proteins that sample inactive, intermediate, and active conformational states characterized by relatively slow interconversions (~µs-ms). On a faster timescale (~ps-ns), the conformational landscape of GPCRs is governed by the rapid dynamics of amino acid side chains. Such dynamics are essential for protein functions such as ligand recognition and allostery. Unfortunately, technical challenges have almost entirely precluded the study of side-chain dynamics for GPCRs. Here, we investigate the rapid side-chain dynamics of a thermostabilized α1B -adrenergic receptor (α1B -AR) as probed by methyl relaxation. We determined order parameters for Ile, Leu, and Val methyl groups in the presence of inverse agonists that bind orthosterically (prazosin, tamsulosin) or allosterically (conopeptide ρ-TIA). Despite the differences in the ligands, the receptor's overall side-chain dynamics are very similar, including those of the apo form. However, ρ-TIA increases the flexibility of Ile1764×56 and possibly of Ile2145×49 , adjacent to Pro2155×50 of the highly conserved P5×50 I3×40 F6×44 motif crucial for receptor activation, suggesting differences in the mechanisms for orthosteric and allosteric receptor inactivation. Overall, increased Ile side-chain rigidity was found for residues closer to the center of the membrane bilayer, correlating with denser packing and lower protein surface exposure. In contrast to two microbial membrane proteins, in α1B -AR Leu exhibited higher flexibility than Ile side chains on average, correlating with the presence of Leu in less densely packed areas and with higher protein-surface exposure than Ile. Our findings demonstrate the feasibility of studying receptor-wide side-chain dynamics in GPCRs to gain functional insights.

Evolution of Cd2+ and Cu+ binding in Helix pomatia metallothioneins.

Metallothioneins (MTs) are small proteins present in all kingdoms of life. Their high cysteine content enables them to bind metal ions, such as Zn2+, Cd2+, and Cu+, providing means for detoxification and metal homeostasis. Three MT isoforms with distinct metal binding preferences are present in the Roman Snail Helix pomatia. Here, we use nuclear magnetic resonance (NMR) to follow the evolution of Cd2+ and Cu+ binding from the reconstructed ancestral Stylommatophora MT to the three H. pomatia MT (HpMT) isoforms. Information obtained from [15N,1H]-HSQC spectra and T2 relaxation times are combined to describe the conformational stability of the MT-metal complexes. A well-behaved MT-metal complex adopts a unique structure and does not undergo additional conformational exchange. The ancestor to all three HpMTs forms conformationally stable Cd2+ complexes and closely resembles the Cd2+-specific HpCdMT isoform, suggesting a role in Cd2+ detoxification for the ancestral protein. All Cu+-MT complexes, including the Cu+-specific HpCuMT isoform, undergo a considerable amount of conformational exchange. The unspecific HpCd/CuMT and the Cu+-specific HpCuMT isoforms form Cu+ complexes with comparable characteristics. It is possible to follow how Cd2+ and Cu+ binding changed throughout evolution. Interestingly, Cu+ binding improved independently in the lineages leading to the unspecific and the Cu+-specific HpMT isoforms. C-terminal domains are generally less capable of coordinating the non-cognate metal ion than N-terminal domains, indicating a higher level of specialization of the C-domain. Our findings provide new insights into snail MT evolution, helping to understand the interplay between biological function and structural features toward a comprehensive understanding of metal preference.

Early Molecular Insights into Thanatin Analogues Binding to A. baumannii LptA.

The cationic antimicrobial ß-hairpin, thanatin, was recently developed into drug-like analogues active against carbapenem-resistant Enterobacteriaceae (CRE). The analogues represent new antibiotics with a novel mode of action targeting LptA in the periplasm and disrupting LPS transport. The compounds lose antimicrobial efficacy when the sequence identity to E. coli LptA falls below 70%. We wanted to test the thanatin analogues against LptA of a phylogenetic distant organism and investigate the molecular determinants of inactivity. Acinetobacter baumannii (A. baumannii) is a critical Gram-negative pathogen that has gained increasing attention for its multi-drug resistance and hospital burden. A. baumannii LptA shares 28% sequence identity with E. coli LptA and displays an intrinsic resistance to thanatin and thanatin analogues (MIC values > 32 µg/mL) through a mechanism not yet described. We investigated the inactivity further and discovered that these CRE-optimized derivatives can bind to LptA of A. baumannii in vitro, despite the high MIC values. Herein, we present a high-resolution structure of A. baumannii LptAm in complex with a thanatin derivative 7 and binding affinities of selected thanatin derivatives. Together, these data offer structural insights into why thanatin derivatives are inactive against A. baumannii LptA, despite binding events in vitro.

Peptidomimetic antibiotics disrupt the lipopolysaccharide transport bridge of drug-resistant Enterobacteriaceae.

The rise of antimicrobial resistance poses a substantial threat to our health system, and, hence, development of drugs against novel targets is urgently needed. The natural peptide thanatin kills Gram-negative bacteria by targeting proteins of the lipopolysaccharide transport (Lpt) machinery. Using the thanatin scaffold together with phenotypic medicinal chemistry, structural data, and a target-focused approach, we developed antimicrobial peptides with drug-like properties. They exhibit potent activity against Enterobacteriaceae both in vitro and in vivo while eliciting low frequencies of resistance. We show that the peptides bind LptA of both wild-type and thanatin-resistant Escherichia coli and Klebsiella pneumoniae strains with low-nanomolar affinities. Mode of action studies revealed that the antimicrobial activity involves the specific disruption of the Lpt periplasmic protein bridge.

Discovery of a small molecule ligand of FRS2 that inhibits invasion and tumor growth.

PURPOSE: Aberrant activation of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases drives oncogenic signaling through its proximal adaptor protein FRS2. Precise disruption of this disease-causing signal transmission in metastatic cancers could stall tumor growth and progression. The purpose of this study was to identify a small molecule ligand of FRS2 to interrupt oncogenic signal transmission from activated FGFRs. METHODS: We used pharmacophore-based computational screening to identify potential small molecule ligands of the PTB domain of FRS2, which couples FRS2 to FGFRs. We confirmed PTB domain binding of molecules identified with biophysical binding assays and validated compound activity in cell-based functional assays in vitro and in an ovarian cancer model in vivo. We used thermal proteome profiling to identify potential off-targets of the lead compound. RESULTS: We describe a small molecule ligand of the PTB domain of FRS2 that prevents FRS2 activation and interrupts FGFR signaling. This PTB-domain ligand displays on-target activity in cells and stalls FGFR-dependent matrix invasion in various cancer models. The small molecule ligand is detectable in the serum of mice at the effective concentration for prolonged time and reduces growth of the ovarian cancer model in vivo. Using thermal proteome profiling, we furthermore identified potential off-targets of the lead compound that will guide further compound refinement and drug development. CONCLUSIONS: Our results illustrate a phenotype-guided drug discovery strategy that identified a novel mechanism to repress FGFR-driven invasiveness and growth in human cancers. The here identified bioactive leads targeting FGF signaling and cell dissemination provide a novel structural basis for further development as a tumor agnostic strategy to repress FGFR- and FRS2-driven tumors.

Improved Repeat Protein Stability by Combined Consensus and Computational Protein Design.

High protein stability is an important feature for proteins used as therapeutics, as diagnostics, and in basic research. We have previously employed consensus design to engineer optimized Armadillo repeat proteins (ArmRPs) for sequence-specific recognition of linear epitopes with a modular binding mode. These designed ArmRPs (dArmRPs) feature high stability and are composed of M-type internal repeats that are flanked by N- and C-terminal capping repeats that protect the hydrophobic core from solvent exposure. While the overall stability of the designed ArmRPs is remarkably high, subsequent biochemical and biophysical experiments revealed that the N-capping repeat assumes a partially unfolded, solvent-accessible conformation for a small fraction of time that renders it vulnerable to proteolysis and aggregation. To overcome this problem, we have designed new N-caps starting from an M-type internal repeat using the Rosetta software. The superior stability of the computationally refined models was experimentally verified by circular dichroism and nuclear magnetic resonance spectroscopy. A crystal structure of a dArmRP containing the novel N-cap revealed that the enhanced stability correlates with an improved packing of this N-cap onto the hydrophobic core of the dArmRP. Hydrogen exchange experiments further show that the level of local unfolding of the N-cap is reduced by several orders of magnitude, resulting in increased resistance to proteolysis and weakened aggregation. As a first application of the novel N-cap, we determined the solution structure of a dArmRP with four internal repeats, which was previously impeded by the instability of the original N-cap.

A conformational switch controlling the toxicity of the prion protein.

Prion infections cause conformational changes of the cellular prion protein (PrPC) and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ('H-latch'), altering the flexibility of the α2-α3 and ß2-α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is an important reporter of prion neurotoxicity.

Crystal structure of the α1B-adrenergic receptor reveals molecular determinants of selective ligand recognition.

α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.

An automated iterative approach for protein structure refinement using pseudocontact shifts.

NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM4A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing.

Peptides in BioNMR Research.

Heteronuclear NMR in combination with isotope labelling is used to study folding of polypeptides induced by metals in the case of metallothioneins, binding of the peptidic allosteric modulator ρ-TIA to the human G-protein coupled α1b adrenergic receptor, the development of therapeutic drugs that interfere with the biosynthesis of the outer membrane of Gram-negative bacteria, and a system in which protein assembly is induced upon peptide addition. NMR in these cases is used to derive precise structural data and to study the dynamics.

Dynamics of Bacteriorhodopsin in the Dark-Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy.

To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side-chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR.

Optimizing the α1B-adrenergic receptor for solution NMR studies.

Sample preparation for NMR studies of G protein-coupled receptors faces special requirements: Proteins need to be stable for prolonged measurements at elevated temperatures, they should ideally be uniformly labeled with the stable isotopes 13C, 15N, and all carbon-bound protons should be replaced by deuterons. In addition, certain NMR experiments require protonated methyl groups in the presence of a perdeuterated background. All these requirements are most easily satisfied when using Escherichia coli as the expression host. Here we describe a workflow, starting from a temperature-stabilized mutant of the α1B-adrenergic receptor, obtained using the CHESS methodology, into an even more stable species, in which flexible parts from termini were removed and the intracellular loop 3 (ICL3) was stabilized against proteolytic cleavage. The yield after purification corresponds to 1-2 mg/L of D2O culture. The final purification step is ligand-affinity chromatography to ensure that only well-folded ligand-binding protein is isolated. Proper selection of detergent has a remarkable influence on the quality of NMR spectra. All optimization steps of sequence and detergent are monitored on a small scale by monitoring the melting temperature and long-term thermal stability to allow for screening of many conditions. The stabilized mutant of the α1B-adrenergic receptor was additionally incorporated in nanodiscs, but displayed slightly inferior spectra compared to a sample in detergent micelles. Finally, both [15N,1H]- as well as [13C,1H]-HSQC spectra are shown highlighting the high quality of the final NMR sample. Importantly, the quality of [13C,1H]-HSQC spectra indicates that the so prepared receptor could be used for studying side-chain dynamics.

Metallomics reveals a persisting impact of cadmium on the evolution of metal-selective snail metallothioneins.

The tiny contribution of cadmium (Cd) to the composition of the earth's crust contrasts with its high biological significance, owing mainly to the competition of Cd with the essential zinc (Zn) for suitable metal binding sites in proteins. In this context it was speculated that in several animal lineages, the protein family of metallothioneins (MTs) has evolved to specifically detoxify Cd. Although the multi-functionality and heterometallic composition of MTs in most animal species does not support such an assumption, there are some exceptions to this role, particularly in animal lineages at the roots of animal evolution. In order to substantiate this hypothesis and to further understand MT evolution, we have studied MTs of different snails that exhibit clear Cd-binding preferences in a lineage-specific manner. By applying a metallomics approach including 74 MT sequences from 47 gastropod species, and by combining phylogenomic methods with molecular, biochemical, and spectroscopic techniques, we show that Cd selectivity of snail MTs has resulted from convergent evolution of metal-binding domains that significantly differ in their primary structure. We also demonstrate how their Cd selectivity and specificity has been optimized by the persistent impact of Cd through 430 million years of MT evolution, modifying them upon lineage-specific adaptation of snails to different habitats. Overall, our results support the role of Cd for MT evolution in snails, and provide an interesting example of a vestigial abiotic factor directly driving gene evolution. Finally, we discuss the potential implications of our findings for studies devoted to the understanding of mechanisms leading to metal specificity in proteins, which is important when designing metal-selective peptides.

Impact of naturally occurring serine/cysteine variations on the structure and function of Pseudomonas metallothioneins.

Metallothioneins (MTs), small cysteine-rich metal-binding proteins, support the viability of organisms under normal physiological conditions and help them to respond to different environmental stressors. Upon metal coordination (e.g. ZnII, CdII, CuI) they form characteristic polynuclear metal-thiolate clusters that are known for their high thermodynamic stability and kinetic lability. However, despite numerous studies, it is still not understood how MTs modulate their metal-binding properties. Pseudomonas MTs are an emerging subclass of bacterial MTs, distinct for their high number of His residues and for several unique features such as an intrinsically disordered long C-terminal tail and multiple variations in the number and nature of coordinating amino acids. These variations might provide the bacteria with a functional advantage derived from evolutionary adaptation to heterogeneous environments. Nearly 90% of the known Pseudomonas MT sequences feature a central YCC[combining low line]xxC motif, that is altered to YCS[combining low line]xxC in the rest. We demonstrate that the additional Cys residue serves as a coordinating ligand without influencing the metal-binding capacity, the overall metal-binding stability or the structure. However, the additional ligand changes intra-cluster dynamics and, as a consequence, modulates metal transfer reactions that could be functionally advantageous in vivo.

Backbone and methyl assignment of bacteriorhodopsin incorporated into nanodiscs.

Resonance assignments are challenging for membrane proteins due to the size of the lipid/detergent-protein complex and the presence of line-broadening from conformational exchange. As a consequence, many correlations are missing in the triple-resonance NMR experiments typically used for assignments. Herein, we present an approach in which correlations from these solution-state NMR experiments are supplemented by data from 13C unlabeling, single-amino acid type labeling, 4D NOESY data and proximity of moieties to lipids or water in combination with a structure of the protein. These additional data are used to edit the expected peaklists for the automated assignment protocol FLYA, a module of the program package CYANA. We demonstrate application of the protocol to the 262-residue proton pump from archaeal bacteriorhodopsin (bR) in lipid nanodiscs. The lipid-protein assembly is characterized by an overall correlation time of 44 ns. The protocol yielded assignments for 62% of all backbone (H, N, Cα, Cß, C') resonances of bR, corresponding to 74% of all observed backbone spin systems, and 60% of the Ala, Met, Ile (δ1), Leu and Val methyl groups, thus enabling to assign a large fraction of the protein without mutagenesis data. Most missing resonances stem from the extracellular half, likely due intermediate exchange line-broadening. Further analysis revealed that missing information of the amino acid type of the preceding residue is the largest problem, and that 4D NOESY experiments are particularly helpful to compensate for that information loss.

Distinct pathways for zinc metabolism in the terrestrial slug Arion vulgaris.

In most organisms, the concentration of free Zn2+ is controlled by metallothioneins (MTs). In contrast, no significant proportions of Zn2+ are bound to MTs in the slug, Arion vulgaris. Instead, this species possesses cytoplasmic low-molecular-weight Zn2+ (LMW Zn) binding compound that divert these metal ions into pathways uncoupled from MT metabolism. Zn2+ is accumulated in the midgut gland calcium cells of Arion vulgaris, where they associate with a low-molecular-weight ligand with an apparent molecular mass of ~ 2,000 Da. Mass spectrometry of the semi-purified LMW Zn binding compound combining an electrospray ion source with a differential mobility analyser coupled to a time-of-flight mass spectrometer revealed the presence of four Zn2+-containing ion signals, which arise from disintegration of one higher MW complex resulting in an ion-mobility diameter of 1.62 nm and a molecular mass of 837 Da. We expect that the novel Zn2+ ion storage pathway may be shared by many other gastropods, and particularly species that possess Cd-selective MT isoforms or variants with only very low affinity to Zn2+.

The Solution Structure and Dynamics of Cd-Metallothionein from Helix pomatia Reveal Optimization for Binding Cd over Zn.

Metallothioneins (MTs) are cysteine-rich polypeptides that are naturally found coordinated to monovalent and/or divalent transition metal ions. Three metallothionein isoforms from the Roman snail Helix pomatia are known. They differ in their physiological metal load and in their specificity for transition metal ions such as Cd2+ (HpCdMT isoform) and Cu+ (HpCuMT isoform) or in the absence of a defined metal specificity (HpCd/CuMT isoform). We have determined the solution structure of the Cd-specific isoform (HpCdMT) by nuclear magnetic resonance spectroscopy using recombinant isotopically labeled protein loaded with Zn2+ or Cd2+. Both structures display two-domain architectures, where each domain comprises a characteristic three-metal cluster similar to that observed in the ß-domains of vertebrate MTs. The polypeptide backbone is well-structured over the entire sequence, including the interdomain linker. Interestingly, the two domains display mutual contacts, as observed before for the metallothionein of the snail Littorina littorea, to which both N- and C-terminal domains are highly similar. Increasing the length of the linker motionally decouples both domains and removes mutual contacts between them without having a strong effect on the stability of the individual domains. The structures of Cd6- and Zn6-HpCdMT are nearly identical. However, 15N relaxation, in particular 15N R2 rates, is accelerated for many residues of Zn6-HpCdMT but not for Cd6-HpCdMT, revealing the presence of conformational exchange effects. We suggest that this snail MT isoform is evolutionarily optimized for binding Cd rather than Zn.