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.

Inositol pyrophosphates activate the vacuolar transport chaperone complex in yeast by disrupting a homotypic SPX domain interaction.

Many proteins involved in eukaryotic phosphate homeostasis are regulated by SPX domains. In yeast, the vacuolar transporter chaperone (VTC) complex contains two such domains, but mechanistic details of its regulation are not well understood. Here, we show at the atomic level how inositol pyrophosphates interact with SPX domains of subunits Vtc2 and Vtc3 to control the activity of the VTC complex. Vtc2 inhibits the catalytically active VTC subunit Vtc4 by homotypic SPX-SPX interactions via the conserved helix α1 and the previously undescribed helix α7. Binding of inositol pyrophosphates to Vtc2 abrogates this interaction, thus activating the VTC complex. Accordingly, VTC activation is also achieved by site-specific point mutations that disrupt the SPX-SPX interface. Structural data suggest that ligand binding induces reorientation of helix α1 and exposes the modifiable helix α7, which might facilitate its post-translational modification in vivo. The variable composition of these regions within the SPX domain family might contribute to the diversified SPX functions in eukaryotic phosphate homeostasis.

Sculpting conducting nanopore size and shape through de novo protein design.

Transmembrane ß-barrels (TMBs) are widely used for single molecule DNA and RNA sequencing and have considerable potential for a broad range of sensing and sequencing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels such as CsgG, which have evolved to carry out functions very different from sensing, and hence provide sub-optimal starting points. In contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to the design of transmembrane ß-barrel pores with different diameter and pore geometry. NMR and crystallographic characterization shows that the designs are stably folded with structures close to the design models. We report the first examples of de novo designed TMBs with 10, 12 and 14 stranded ß-barrels. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 pS (~0.5 nm pore diameter) to 430 pS (~1.1 nm pore diameter), and can be converted into sensitive small-molecule sensors with high signal to noise ratio. The capability to generate on demand ß-barrel pores of defined geometry opens up fundamentally new opportunities for custom engineering of sequencing and sensing technologies.

Pseudocontact Shifts in Biomolecular NMR Spectroscopy.

Paramagnetic centers in biomolecules, such as specific metal ions that are bound to a protein, affect the nuclei in their surrounding in various ways. One of these effects is the pseudocontact shift (PCS), which leads to strong chemical shift perturbations of nuclear spins, with a remarkably long range of 50 Å and beyond. The PCS in solution NMR is an effect originating from the anisotropic part of the dipole-dipole interaction between the magnetic momentum of unpaired electrons and nuclear spins. The PCS contains spatial information that can be exploited in multiple ways to characterize structure, function, and dynamics of biomacromolecules. It can be used to refine structures, magnify effects of dynamics, help resonance assignments, allows for an intermolecular positioning system, and gives structural information in sensitivity-limited situations where all other methods fail. Here, we review applications of the PCS in biomolecular solution NMR spectroscopy, starting from early works on natural metalloproteins, following the development of non-natural tags to chelate and attach lanthanoid ions to any biomolecular target to advanced applications on large biomolecular complexes and inside living cells. We thus hope to not only highlight past applications but also shed light on the tremendous potential the PCS has in structural biology.

Bicyclic Phenyl-Ethynyl Architectures: Synthesis of a 1,4-Bis(phenylbuta-1,3-diyn-1-yl) Benzene Banister.

The novel diacetylene bridged terphenylic macrocycle 1 is presented and discussed in the context of rotationally restricted "Geländer" oligomers. The 1,4-bis(phenylbuta-1,3-diyn-1-yl) benzene bridge of diacetylene 1 is significantly longer than its terphenyl backbone, forcing the bridge to bend around the central pylon. The synthesis of molecule 1 is based to a large extent on acetylene scaffolding strategies, profiting from orthogonal alkyne protection groups to close both macrocyclic subunits by oxidative acetylene coupling sequentially. The spatial arrangement and the dynamic enantiomerization process of the bicyclic target structure 1 are analyzed. In-depth NMR investigations not only reveal an unexpected spatial arrangement with both oligomer strands bent alongside the backbone, but also display the limited stability of the model compound in the presence of molecular oxygen.

NMR pseudocontact shifts in a symmetric protein homotrimer.

NMR pseudocontact shifts are a valuable tool for structural and functional studies of proteins. Protein multimers mediate key functional roles in biology, but methods for their study by pseudocontact shifts are so far not available. Paramagnetic tags attached to identical subunits in multimeric proteins cause a combined pseudocontact shift that cannot be described by the standard single-point model. Here, we report pseudocontact shifts generated simultaneously by three paramagnetic Tm-M7PyThiazole-DOTA tags to the trimeric molecular chaperone Skp and provide an approach for the analysis of this and related symmetric systems. The pseudocontact shifts were described by a "three-point" model, in which positions and parameters of the three paramagnetic tags were fitted. A good correlation between experimental data and predicted values was found, validating the approach. The study establishes that pseudocontact shifts can readily be applied to multimeric proteins, offering new perspectives for studies of large protein complexes by paramagnetic NMR spectroscopy.

Localization of ligands within human carbonic anhydrase II using 19F pseudocontact shift analysis.

Unraveling the native structure of protein-ligand complexes in solution enables rational drug design. We report here the use of 19F pseudocontact shift (PCS) NMR as a method to determine fluorine positions of high affinity ligands bound within the drug target human carbonic anhydrase II with high accuracy. Three different ligands were localized within the protein by analysis of the obtained PCS from simple one-dimensional 19F spectra with an accuracy of up to 0.8 Å. In order to validate the PCS, four to five independent magnetic susceptibility tensors induced by lanthanide chelating tags bound site-specifically to single cysteine mutants were refined. Least-squares minimization and a Monte-Carlo approach allowed the assessment of experimental errors on the intersection of the corresponding four to five PCS isosurfaces. By defining an angle score that reflects the relative isosurface orientation for different tensor combinations, it was established that the ligand can be localized accurately using only three tensors, if the isosurfaces are close to orthogonal. For two out of three ligands, the determined position closely matched the X-ray coordinates. Our results for the third ligand suggest, in accordance with previously reported ab initio calculations, a rotated position for the difluorophenyl substituent, enabling a favorable interaction with Phe-131. The lanthanide-fluorine distance varied between 22 and 38 Å and induced 19F PCS ranged from 0.078 to 0.409 ppm, averaging to 0.213 ppm. Accordingly, even longer metal-fluorine distances will lead to meaningful PCS, rendering the investigation of protein-ligand complexes significantly larger than 30 kDa feasible.

Synthesis of chiral nine and twelve-membered cyclic polyamines from natural building blocks.

A rational strategy for the facile and efficient cyclization of amino acid-based linear precursors forming nine and twelve-membered cyclic peptidomimetics is reported. The resulting chiral lactams can readily be reduced to substituted cyclic polyamine analogues of 1,4,7,10-tetraaza-cyclododecane (cyclen) and 1,4,7-triaza-cyclononane (TACN).

New Lanthanide Chelating Tags for PCS NMR Spectroscopy with Reduction Stable, Rigid Linkers for Fast and Irreversible Conjugation to Proteins.

Lanthanide chelating tags (LCTs) have been used with great success for determining structures and interactions of proteins and other biological macromolecules. Recently LCTs have also been used for in-cell NMR spectroscopy, but the bottleneck especially for demanding applications like pseudocontact shift (PCS) NMR is the sparse availability of suitable tags that allow for site-selective, rigid, irreversible, fast, and quantitative conjugation of chelated paramagnetic lanthanide ions to proteins via reduction stable bonds. We report here several such tags and focus on a new pyridine thiazole derivate of DOTA, that combines high affinity, rigidity, and selectivity with unprecedented tagging properties. The conjugation to the cysteine thiol of the protein results in a reductively stable thioether bond and proceeds virtually quantitatively in less than 30 min at 100 µM protein concentration, ambient temperature, and neutral pH. Upon conjugation of the new tag to two single cysteine mutants of ubiquitin and a single cysteine mutant of human carbonic anhydrase type II (30 kDa) only one stereoisomer is formed (square antiprismatic coordination, Λ(δδδδ)) and large to very large pseudocontact shifts as well as large residual dipolar couplings (RDCs) are observed by NMR spectroscopy. The PCS and RDC show excellent agreement with the solid state structure of the proteins. We believe that the pyridine thiazole moiety reported here has the potential to serve as a thiole reactive group in various conjugation applications; furthermore, its terbium complex shows strong photoluminescence upon irradiation and may thus serve as a donor group for Förster resonance energy transfer spectroscopy.

Synthesis of trinorbornane.

Herein we report the synthesis and characterisation of the until recently unreported chiral C11 skeleton of tetracyclo[5.2.2.01,6.04,9]undecane ("trinorbornane") which could be obtained in 7% overall yield in 9 steps. This new rigid structural type was found to be present in the computer generated Chemical Universe Data-base (GDB) and has until now no real-world counterpart.

In-Cell Protein Structures from 2D NMR Experiments.

In-cell NMR spectroscopy provides atomic resolution insights into the structural properties of proteins in cells, but it is rarely used to solve entire protein structures de novo. Here, we introduce a paramagnetic lanthanide-tag to simultaneously measure protein pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) to be used as input for structure calculation routines within the Rosetta program. We employ this approach to determine the structure of the protein G B1 domain (GB1) in intact Xenopus laevis oocytes from a single set of 2D in-cell NMR experiments. Specifically, we derive well-defined GB1 ensembles from low concentration in-cell NMR samples (∼50 µM) measured at moderate magnetic field strengths (600 MHz), thus offering an easily accessible alternative for determining intracellular protein structures.