Paramagnetic Chemical Probes for Studying Biological Macromolecules.

Paramagnetic chemical probes have been used in electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy for more than four decades. Recent years witnessed a great increase in the variety of probes for the study of biological macromolecules (proteins, nucleic acids, and oligosaccharides). This Review aims to provide a comprehensive overview of the existing paramagnetic chemical probes, including chemical synthetic approaches, functional properties, and selected applications. Recent developments have seen, in particular, a rapid expansion of the range of lanthanoid probes with anisotropic magnetic susceptibilities for the generation of structural restraints based on residual dipolar couplings and pseudocontact shifts in solution and solid state NMR spectroscopy, mostly for protein studies. Also many new isotropic paramagnetic probes, suitable for NMR measurements of paramagnetic relaxation enhancements, as well as EPR spectroscopic studies (in particular double resonance techniques) have been developed and employed to investigate biological macromolecules. Notwithstanding the large number of reported probes, only few have found broad application and further development of probes for dedicated applications is foreseen.

Rigidified and Hydrophilic DOTA-like Lanthanoid Ligands: Design, Synthesis, and Dynamic Properties.

Limiting the dynamics of paramagnetic tags is crucial for the accuracy of the structural information derived from paramagnetic nuclear magnetic resonance (NMR) experiments. A hydrophilic rigid 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA)-like lanthanoid complex was designed and synthesized following a strategy that allows the incorporation of two sets of two adjacent substituents. This resulted in a C2 symmetric hydrophilic and rigid macrocyclic ring, featuring four chiral hydroxyl-methylene substituents. NMR spectroscopy was used to investigate the conformational dynamics of the novel macrocycle upon complexation with europium and compared to DOTA and its derivatives. The twisted square antiprismatic and square antiprismatic conformers coexist, but the former is favored, which is different from DOTA. Two-dimensional 1H exchange spectroscopy shows that ring flipping of the cyclen-ring is suppressed due to the presence of the four chiral equatorial hydroxyl-methylene substituents at proximate positions. The reorientation of the pendant arms causes conformational exchange between two conformers. The reorientation of the coordination arms is slower when the ring flipping is suppressed. This indicates that these complexes are suitable scaffolds to develop rigid probes for paramagnetic NMR of proteins. Due to their hydrophilic nature, it is anticipated that they are less likely to cause protein precipitation than their more hydrophobic counterparts.

Novel anti-repression mechanism of H-NS proteins by a phage protein.

H-NS family proteins, bacterial xenogeneic silencers, play central roles in genome organization and in the regulation of foreign genes. It is thought that gene repression is directly dependent on the DNA binding modes of H-NS family proteins. These proteins form lateral protofilaments along DNA. Under specific environmental conditions they switch to bridging two DNA duplexes. This switching is a direct effect of environmental conditions on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of H-NS proteins. The Pseudomonas lytic phage LUZ24 encodes the protein gp4, which modulates the DNA binding and function of the H-NS family protein MvaT of Pseudomonas aeruginosa. However, the mechanism by which gp4 affects MvaT activity remains elusive. In this study, we show that gp4 specifically interferes with the formation and stability of the bridged MvaT-DNA complex. Structural investigations suggest that gp4 acts as an 'electrostatic zipper' between the oppositely charged domains of MvaT protomers, and stabilizes a structure resembling their 'half-open' conformation, resulting in relief of gene silencing and adverse effects on P. aeruginosa growth. The ability to control H-NS conformation and thereby its impact on global gene regulation and growth might open new avenues to fight Pseudomonas multidrug resistance.

Structural basis for osmotic regulation of the DNA binding properties of H-NS proteins.

H-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive. Here, we focus on the H-NS family protein MvaT from Pseudomonas aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strength on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.

The Charge Distribution on a Protein Surface Determines Whether Productive or Futile Encounter Complexes Are Formed.

Protein complex formation depends strongly on electrostatic interactions. The distribution of charges on the surface of redox proteins is often optimized by evolution to guide recognition and binding. To test the degree to which the electrostatic interactions between cytochrome c peroxidase (CcP) and cytochrome c (Cc) are optimized, we produced five CcP variants, each with a different charge distribution on the surface. Monte Carlo simulations show that the addition of negative charges attracts Cc to the new patches, and the neutralization of the charges in the regular, stereospecific binding site for Cc abolishes the electrostatic interactions in that region entirely. For CcP variants with the charges in the regular binding site intact, additional negative patches slightly enhance productive complex formation, despite disrupting the optimized charge distribution. Removal of the charges in the regular binding site results in a dramatic decrease in the complex formation rate, even in the presence of highly negative patches elsewhere on the surface. We conclude that additional charge patches can result in either productive or futile encounter complexes, depending on whether negative residues are located also in the regular binding site.

The G132S Mutation Enhances the Resistance of Mycobacterium tuberculosis ß-Lactamase against Sulbactam.

The current rise of antibiotic resistant forms of Mycobacterium tuberculosis is a global health threat that calls for new antibiotics. The ß-lactamase BlaC of this pathogen prevents the use of ß-lactam antibiotics, except in combination with a ß-lactamase inhibitor. To understand if exposure to such inhibitors can easily result in resistance, a BlaC evolution experiment was performed, studying the evolutionary adaptability against the inhibitor sulbactam. Several amino acid substitutions in BlaC were shown to confer reduced sensitivity to sulbactam. The G132S mutation causes a reduction in the rate of nitrocefin and ampicillin hydrolysis and simultaneously reduces the sensitivity for sulbactam inhibition. Introduction of the side chain moiety of Ser132 causes the 104-105 peptide bond to assume the cis conformation and the side chain of Ser104 to be rotated toward the sulbactam adduct with which it forms a hydrogen bond not present in the wild-type enzyme. The gatekeeper residue Ile105 also moves. These changes in the entrance of the active site can explain the decreased affinity of G132S BlaC for both substrates and sulbactam. Our results show that BlaC can easily acquire a reduced sensitivity for sulbactam, with a single-amino acid mutation, which could hinder the use of combination therapies.

Partial Opening of Cytochrome P450cam (CYP101A1) Is Driven by Allostery and Putidaredoxin Binding.

Cytochrome P450cam (CYP101A1) catalyzes the regio- and stereo-specific 5-exo-hydroxylation of camphor via a multistep catalytic cycle that involves two-electron transfer steps, with an absolute requirement that the second electron be donated by the ferrodoxin, putidaredoxin (Pdx). Whether P450cam, once camphor has bound to the active site and the substrate entry channel has closed, opens up upon Pdx binding, during the second electron transfer step, or it remains closed is still a matter of debate. A potential allosteric site for camphor binding has been identified and postulated to play a role in the binding of Pdx. Here, we have revisited paramagnetic NMR spectroscopy data and determined a heterogeneous ensemble of structures that explains the data, provides a complete representation of the P450cam/Pdx complex in solution, and reconciles alternative hypotheses. The allosteric camphor binding site is always present, and the conformational changes induced by camphor binding to this site facilitates Pdx binding. We also determined that the state to which Pdx binds comprises an ensemble of structures that have features of both the open and closed state. These results demonstrate that there is a finely balanced interaction between allosteric camphor binding and the binding of Pdx at high camphor concentrations.

Protein Dynamics Influence the Enzymatic Activity of Phospholipase A/Acyltransferases 3 and 4.

Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bioactive lipids. Despite sequential and structural similarities, the two enzymes differ in activity and specificity. The relation between the activity and dynamics of the N-terminal domains of PLAAT3 and PLAAT4 was studied. PLAAT3 has a much higher melting temperature and exhibits less nanosecond and millisecond dynamics in the active site, in particular in loop L2(B6), as shown by NMR spectroscopy and molecular dynamics calculations. Swapping the L2(B6) loops between the two PLAAT enzymes results in strongly increased phospholipase activity in PLAAT3 but no reduction in PLAAT4 activity, indicating that this loop contributes to the low activity of PLAAT3. The results show that, despite structural similarity, protein dynamics differ substantially between the PLAAT variants, which can help to explain the activity and specificity differences.

Two ß-Lactamase Variants with Reduced Clavulanic Acid Inhibition Display Different Millisecond Dynamics.

The ß-lactamase of Mycobacterium tuberculosis, BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli. The variant that was found to confer the most inhibitor resistance, K234R, as well as variant G132N that was found previously were characterized using X-ray crystallography and nuclear magnetic resonance (NMR) relaxation experiments to probe structural and dynamic properties. The G132N mutant exists in solution in two almost equally populated conformations that exchange with a rate of ca. 88 s-1. The conformational change affects a broad region of the enzyme. The crystal structure reveals that the Asn132 side chain forces the peptide bond between Ser104 and Ile105 in a cis-conformation. The crystal structure suggests multiple conformations for several side chains (e.g., Ser104 and Ser130) and a short loop (positions 214 to 216). In the K234R mutant, the active-site dynamics are significantly diminished with respect to the wild-type enzyme. These results show that multiple evolutionary routes are available to increase inhibitor resistance in BlaC and that active-site dynamics on the millisecond time scale are not required for catalytic function.

Tuning the Transglycosylation Reaction of a GH11 Xylanase by a Delicate Enhancement of its Thumb Flexibility.

Glycoside hydrolases (GHs) are attractive tools for multiple biotechnological applications. In conjunction with their hydrolytic function, GHs can perform transglycosylation under specific conditions. In nature, oligosaccharide synthesis is performed by glycosyltransferases (GTs); however, the industrial use of GTs is limited by their instability in solution. A key difference between GTs and GHs is the flexibility of their binding site architecture. We have used the xylanase from Bacillus circulans (BCX) to study the interplay between active-site flexibility and transglycosylation. Residues of the BCX "thumb" were substituted to increase the flexibility of the enzyme binding site. Replacement of the highly conserved residue P116 with glycine shifted the balance of the BCX enzymatic reaction toward transglycosylation. The effects of this point mutation on the structure and dynamics of BCX were investigated by NMR spectroscopy. The P116G mutation induces subtle changes in the configuration of the thumb and enhances the millisecond dynamics of the active site. Based on our findings, we propose the remodelling of the GH enzymes glycon site flexibility as a strategy to improve the transglycosylation efficiency of these biotechnologically important catalysts.

The Resting Oxidized State of Small Laccase Analyzed with Paramagnetic NMR Spectroscopy.

The enzyme laccase catalyzes the reduction of dioxygen to water at the trinuclear copper center (TNC). The TNC comprises a type-3 (T3) and a type-2 (T2) copper site. The paramagnetic NMR spectrum of the small laccase from Streptomyces coelicolor (SLAC) without the substrate shows a mixture of two catalytic states, the resting oxidized (RO) state and the native intermediate (NI) state. An analysis of the resonances of the RO state is reported. In this state, hydrogen resonances only of the T3 copper ligands can be found, in the region of 12-22 ppm. Signals from all six histidine ligands are found and can be attributed to Hδ1, Hß or backbone amide HN nuclei. Two sequence-specific assignments are proposed on the basis of a second-coordination shell variant that also lacks the copper ion at the T1 site, SLAC-T1D/Q291E. This double mutant is found to be exclusively in the RO state, revealing a subtle balance between the RO and the NI states.

Dipolar dephasing for structure determination in a paramagnetic environment.

We demonstrate the efficacy of the REDOR-type sequences in determining dipolar coupling strength in a paramagnetic environment. Utilizing paramagnetic effects of enhanced relaxation rates and rapid electronic fluctuations in Cu(II)-(DL-Ala)2.H2O, the dipolar coupling for the methyl C-H that is 4.20 â€‹Å (methyl carbon) away from the Cu2+ ion, was estimated to be 8.8 â€‹± â€‹0.6 â€‹kHz. This coupling is scaled by a factor of ~0.3 in comparison to the rigid limit value of ~32 â€‹kHz, in line with partial averaging of the dipolar interaction by rotational motion of the methyl group. Limited variation in the scaling factor of the dipolar coupling strength at different temperatures is observed. The C-H internuclear distance derived from the size of the dipolar coupling is similar to that observed in the crystal structure. The errors in the dipolar coupling strength observed in the REDOR-type experiments are similar to those reported for diamagnetic systems. Increase in resolution due to the Fermi contact shifts, coupled with MAS frequencies of 30-35 â€‹kHz allowed to estimate the hyperfine coupling strengths for protons and carbons from the temperature dependence of the chemical shift and obtain a high resolution 1H-1H spin diffusion spectrum. This study shows the utility of REDOR-type sequences in obtaining reliable structural and dynamical information from a paramagnetic complex. We believe that this can help in studying the active site of paramagnetic metalloproteins at high resolution.

Chemical Exchange at the Trinuclear Copper Center of Small Laccase from Streptomyces coelicolor.

The trinuclear copper center (TNC) of laccase reduces oxygen to water with very little overpotential. The arrangement of the coppers and ligands in the TNC is known to be from many crystal structures, yet information about possible dynamics of the ligands is absent. Here, we report dynamics at the TNC of small laccase from Streptomyces coelicolor using paramagnetic NMR and electron paramagnetic resonance spectroscopy. Fermi contact-shifted resonances tentatively assigned to histidine Hδ1 display a two-state chemical exchange with exchange rates in the order of 100 s-1. In the electron paramagnetic resonance spectra, at least two forms are observed with different gz-values. It is proposed that the exchange processes reflect the rotational motion of histidine imidazole rings that coordinate the coppers in the TNC.

ß-Lactamase of Mycobacterium tuberculosis Shows Dynamics in the Active Site That Increase upon Inhibitor Binding.

The Mycobacterium tuberculosis ß-lactamase BlaC is a broad-spectrum ß-lactamase that can convert a range of ß-lactam antibiotics. Enzymes with low specificity are expected to exhibit active-site flexibility. To probe the motions in BlaC, we studied the dynamic behavior in solution using nuclear magnetic resonance (NMR) spectroscopy. 15N relaxation experiments show that BlaC is mostly rigid on the pico- to nanosecond timescale. Saturation transfer experiments indicate that also on the high-millisecond timescale BlaC is not dynamic. Using relaxation dispersion experiments, clear evidence was obtained for dynamics in the low-millisecond range, with an exchange rate of ca. 860 s-1 The dynamic amide groups are localized in the active site. Upon formation of an adduct with the inhibitor avibactam, extensive line broadening occurs, indicating an increase in magnitude of the active-site dynamics. Furthermore, the rate of the motions increases significantly. Upon reaction with the inhibitor clavulanic acid, similar line broadening is accompanied by duplication of NMR signals, indicative of at least one additional, slower exchange process (exchange rate, kex, of <100 s-1), while for this inhibitor also loss of pico- to nanosecond timescale rigidity is observed for some amides in the α domain. Possible sources of the observed dynamics, such as motions in the omega loop and rearrangements of active-site residues, are discussed. The increase in dynamics upon ligand binding argues against a model of inhibitor binding through conformational selection. Rather, the induced dynamics may serve to maximize the likelihood of sampling the optimal conformation for hydrolysis of the bound ligand.

A Two-Armed Probe for In-Cell DEER Measurements on Proteins*.

The application of double electron-electron resonance (DEER) with site-directed spin labeling (SDSL) to measure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two-armed GdIII complexes, GdIII -CLaNP13a/b/c were synthesized. Rather than the disulfide linkage of most other CLaNP molecules, a thioether linkage was used to avoid reductive dissociation of the linker. The doubly GdIII labeled N55C/V57C/K147C/T151C variants of T4Lysozyme were measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent with results from paramagnetic NMR. A narrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites.

The Transient Complex of Cytochrome c and Cytochrome c Peroxidase: Insights into the Encounter Complex from Multifrequency EPR and NMR Spectroscopy.

We present a novel approach to study transient protein-protein complexes with standard, 9 GHz, and high-field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso-1-cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate] attached at position 81 of Cc (SL-Cc). A dissociation constant KD of 20±4×10-6  M (EPR and NMR) and an equal amount of stereo-specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc-interaction surface, with the stereo-specific complex.

Identification of productive and futile encounters in an electron transfer protein complex.

Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble of more dynamic orientations: the encounter states. The structure of the stereospecific complex between cytochrome P450cam and putidaredoxin was solved recently by X-ray diffraction as well as paramagnetic NMR spectroscopy. Other than the stereospecific complex, the NMR data clearly show the presence of additional states in the complex in solution. In these encounter states, populated for a small percentage of the time, putidaredoxin assumes multiple orientations and samples a large part of the surface of cytochrome P450cam. To characterize the nature of the encounter states, an extensive paramagnetic NMR dataset has been analyzed using the Maximum Occurrence of Regions methodology. The analysis reveals the location and maximal spatial extent of the additional states needed to fully explain the NMR data. Under the assumption of sparsity of the size of the conformational ensemble, several minor states can be located quite precisely. The distribution of these minor states correlates with the electrostatic potential map around cytochrome P450cam. Whereas some minor states are on isolated positively charged patches, others are connected to the stereospecific site via positively charged paths. The existence of electrostatically favorable pathways between the stereospecific interaction site and the different minor states or lack thereof suggests a means to discriminate between productive and futile encounter states.

Efficient Encounter Complex Formation and Electron Transfer to Cytochrome†c Peroxidase with an Additional, Distant Electrostatic Binding Site.

Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochrome c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner, cytochrome c, interacts with the new patch. Unexpectedly, the rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimized charge distribution.

Dynamics of Ligand Binding to a Rigid Glycosidase*.

The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme-induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction.

New Conformations of Acylation Adducts of Inhibitors of ß-Lactamase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to ß-lactam antibiotics due to the production of the extended spectrum ß-lactamase BlaC. ß-Lactam/ß-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the ß-lactamase inhibitors currently approved for clinical use, clavulanic acid, sulbactam, tazobactam, and avibactam. Here, we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors. The +70 Da adduct derived from clavulanate and the trans-enamine acylation adducts of sulbactam and tazobactam are reported. BlaC in complex with avibactam revealed two inhibitor conformations. Preacylation binding could not be observed because inhibitor binding was not detected in BlaC variants carrying a substitution of the active site serine 70 to either alanine or cysteine, by crystallography, ITC or NMR. These results suggest that the catalytic serine 70 is necessary not only for enzyme acylation but also for increasing BlaC affinity for inhibitors in the preacylation state. The structure of BlaC with the serine to cysteine mutation showed a covalent linkage of the cysteine 70 Sγ atom to the nearby amino group of lysine 73. The differences of adduct conformations between BlaC and other ß-lactamases are discussed.