Enhanced activity against a third-generation cephalosporin by destabilization of the active site of a class A beta-lactamase.

The ß-lactamase BlaC conveys resistance to a broad spectrum of ß-lactam antibiotics to its host Mycobacterium tuberculosis but poorly hydrolyzes third-generation cephalosporins, such as ceftazidime. Variants of other ß-lactamases have been reported to gain activity against ceftazidime at the cost of the native activity. To understand this trade-off, laboratory evolution was performed, screening for enhanced ceftazidime activity. The variant BlaC Pro167Ser shows faster breakdown of ceftazidime, poor hydrolysis of ampicillin and only moderately reduced activity against nitrocefin. NMR spectroscopy, crystallography and kinetic assays demonstrate that the resting state of BlaC P167S exists in an open and a closed state. The open state is more active in the hydrolysis of ceftazidime. In this state the catalytic residue Glu166, generally believed to be involved in the activation of the water molecule required for deacylation, is rotated away from the active site, suggesting it plays no role in the hydrolysis of ceftazidime. In the closed state, deacylation of the BlaC-ceftazidime adduct is slow, while hydrolysis of nitrocefin, which requires the presence of Glu166 in the active site, is barely affected, providing a structural explanation for the trade-off in activities.

Asp179 in the class A ß-lactamase from Mycobacterium tuberculosis is a conserved yet not essential residue due to epistasis.

Conserved residues are often considered essential for function, and substitutions in such residues are expected to have a negative influence on the properties of a protein. However, mutations in a few highly conserved residues of the ß-lactamase from Mycobacterium tuberculosis, BlaC, were shown to have no or only limited negative effect on the enzyme. One such mutant, D179N, even conveyed increased ceftazidime resistance upon bacterial cells, while displaying good activity against penicillins. The crystal structures of BlaC D179N in resting state and in complex with sulbactam reveal subtle structural changes in the Ω-loop as compared to the structure of wild-type BlaC. Introducing this mutation in four other ß-lactamases, CTX-M-14, KPC-2, NMC-A and TEM-1, resulted in decreased antibiotic resistance for penicillins and meropenem. The results demonstrate that the Asp in position 179 is generally essential for class A ß-lactamases but not for BlaC, which can be explained by the importance of the interaction with the side chain of Arg164 that is absent in BlaC. It is concluded that Asp179 though conserved is not essential in BlaC, as a consequence of epistasis.

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.

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.

Mechanical and structural properties of archaeal hypernucleosomes.

Many archaea express histones, which organize the genome and play a key role in gene regulation. The structure and function of archaeal histone-DNA complexes remain however largely unclear. Recent studies show formation of hypernucleosomes consisting of DNA wrapped around an 'endless' histone-protein core. However, if and how such a hypernucleosome structure assembles on a long DNA substrate and which interactions provide for its stability, remains unclear. Here, we describe micromanipulation studies of complexes of the histones HMfA and HMfB with DNA. Our experiments show hypernucleosome assembly which results from cooperative binding of histones to DNA, facilitated by weak stacking interactions between neighboring histone dimers. Furthermore, rotational force spectroscopy demonstrates that the HMfB-DNA complex has a left-handed chirality, but that torque can drive it in a right-handed conformation. The structure of the hypernucleosome thus depends on stacking interactions, torque, and force. In vivo, such modulation of the archaeal hypernucleosome structure may play an important role in transcription regulation in response to environmental changes.

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.

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.

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 Double-Armed, Hydrophilic Transition Metal Complex as a Paramagnetic NMR Probe.

Synthetic metal complexes can be used as paramagnetic probes for the study of proteins and protein complexes. Herein, two transition metal NMR probes (TraNPs) are reported. TraNPs are attached through two arms to a protein to generate a pseudocontact shift (PCS) using cobalt(II), or paramagnetic relaxation enhancement (PRE) with manganese(II). The PCS analysis of TraNPs attached to three different proteins shows that the size of the anisotropic component of the magnetic susceptibility depends on the probe surroundings at the surface of the protein, contrary to what is observed for lanthanoid-based probes. The observed PCS are relatively small, making cobalt-based probes suitable for localized studies, such as of an active site. The obtained PREs are stronger than those obtained with nitroxide spin labels and the possibility to generate both PCS and PRE offers advantages. The properties of TraNPs in comparison with other cobalt-based probes are discussed.

Methyl group reorientation under ligand binding probed by pseudocontact shifts.

Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1-3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.

Phosphate Promotes the Recovery of Mycobacterium tuberculosis ß-Lactamase from Clavulanic Acid Inhibition.

The rise of multi- and even totally antibiotic resistant forms of Mycobacterium tuberculosis underlines the need for new antibiotics. The pathogen is resistant to ß-lactam compounds due to its native serine ß-lactamase, BlaC. This resistance can be circumvented by administration of a ß-lactamase inhibitor. We studied the interaction between BlaC and the inhibitor clavulanic acid. Our data show hydrolysis of clavulanic acid and recovery of BlaC activity upon prolonged incubation. The rate of clavulanic acid hydrolysis is much higher in the presence of phosphate ions. A specific binding site for phosphate is identified in the active site pocket, both in the crystalline state and in solution. NMR spectroscopy experiments show that phosphate binds to this site with a dissociation constant of 30 mM in the free enzyme. We conclude that inhibition of BlaC by clavulanic acid is reversible and that phosphate ions can promote the hydrolysis of the inhibitor.

Methyl group assignment using pseudocontact shifts with PARAssign.

A new version of the program PARAssign has been evaluated for assignment of NMR resonances of the 76 methyl groups in leucines, isoleucines and valines in a 25 kDa protein, using only the structure of the protein and pseudocontact shifts (PCS) generated with a lanthanoid tag at up to three attachment sites. The number of reliable assignments depends strongly on two factors. The principle axes of the magnetic susceptibility tensors of the paramagnetic centers should not be parallel so as to avoid correlated PCS. Second, the fraction of resonances in the spectrum of a paramagnetic sample that can be paired with the diamagnetic counterparts is critical for the assignment. With the data from two tag positions a reliable assignment could be obtained for 60% of the methyl groups and for many of the remaining resonances the number of possible assignments is limited to two or three. With a single tag, reliable assignments can be obtained for methyl groups with large PCS near the tag. It is concluded that assignment of methyl group resonances by paramagnetic tagging can be particularly useful in combination with some additional data, such as from mutagenesis or NOE-based experiments. Approaches to yield the best assignment results with PCS generating tags are discussed.

Delicate conformational balance of the redox enzyme cytochrome P450cam.

The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution. This result contrasts with recent crystal structures of the complex, which suggest that the enzyme opens up when bound to its partner. The closed state supports a model of catalysis in which the substrate is locked in the active site pocket and the enzyme acts as an insulator for the reactive intermediates of the reaction.

A two-armed lanthanoid-chelating paramagnetic NMR probe linked to proteins via thioether linkages.

Paramagnetic NMR probes provide valuable long-range structural information on proteins and protein complexes. A new, stable, two-armed lanthanoid probe is reported that can be attached to a protein site-specifically via chemically inert thioether linkages.

An ensemble of rapidly interconverting orientations in electrostatic protein-peptide complexes characterized by NMR spectroscopy.

Protein complex formation involves an encounter state in which the proteins are associated in a nonspecific manner and often stabilized by interactions between charged surface patches. Such patches are thought to bind in many different orientations with similar affinity. To obtain experimental evidence for the dynamics in encounter complexes, a model was created using the electron transfer protein plastocyanin and short charged peptides. Three plastocyanins with distinct surface charge distributions were studied. The experimental results from chemical shift perturbations, paramagnetic relaxation enhancement (PRE) NMR, and theoretical results from Monte Carlo simulations indicate the presence of multiple binding orientations that interconvert quickly and are dominated by long-range charge interactions. The PRE data also suggest the presence of highly transient orientations stabilized by short-range interactions.

Loss of electrostatic interactions causes increase of dynamics within the plastocyanin-cytochrome f complex.

Recent studies on the electron transfer complex formed by cytochrome f and plastocyanin from Nostoc revealed that both hydrophobic and electrostatic interactions play a role in the process of complex formation. To study the balance between these two types of interactions in the encounter and the final state, the complex between plastocyanin from Phormidium laminosum and cytochrome f from Nostoc sp. PCC 7119 was investigated using NMR spectroscopy and Monte Carlo docking. Cytochrome f has a highly negative charge. Phormidium plastocyanin is similar to that from Nostoc, but the net charge of the protein is negative rather than positive. NMR titrations of Zn-substituted Phormidium plastocyanin and Nostoc cytochrome f indicated that a complex with an affinity intermediate between those of the Nostoc and Phormidium complexes is formed. Plastocyanin was found in a head-on orientation, as determined using pseudocontact shifts, similar to that in the Phormidium complex, in which the hydrophobic patch represents the main site of interaction on plastocyanin. However, the interaction in the cross-complex is dependent on electrostatics, similar to that in the Nostoc complex. The negative charge of plastocyanin decreases, but not abolishes, the attraction to cytochrome f, resulting in the formation of a more diffuse encounter complex than in the Nostoc case, as could be determined using paramagnetic relaxation spectroscopy. This work illustrates the subtle interplay of electrostatic and hydrophobic interactions in the formation of transient protein complexes. The results are discussed in the context of a model for association on the basis of hydrophobic contacts in the encounter state.

Manufacturing of peptides exhibiting biological activity.

Numerous studies have shown that food proteins may be a source of bioactive peptides. Those peptides are encrypted in the protein sequence. They stay inactive within the parental protein until release by proteolytic enzymes (Mine and Kovacs-Nolan in Worlds Poult Sci J 62(1):87-95, 2006; Hartman and Miesel in Curr Opin Biotechnol 18:163-169, 2007). Once released the bioactive peptides exhibit several biofunctionalities and may serve therapeutic roles in body systems. Opioid peptides, peptides lowering high blood pressure, inhibiting platelet aggregation as well as being carriers of metal ions and peptides with immunostimulatory, antimicrobial and antioxidant activities have been described (Hartman and Miesel in Curr Opin Biotechnol 18:163-169, 2007). The biofunctional abilities of the peptides have therefore aroused a lot of scientific, technological and consumer interest with respect to the role of dietary proteins in controlling and influencing health (Möller et al. in Eur J Nutr 47(4):171-182, 2008). Biopeptides may find wide application in food production, the cosmetics industry as well as in the prevention and treatment of various medical conditions. They are manufactured by chemical and biotechnological methods (Marx in Chem Eng News 83(11):17-24. 2005; Hancock and Sahl in Nat Biotechnol 24(12):1551-1557, 2006). Depending on specific needs (food or pharmaceutical industry) different degrees of peptide purifications are required. This paper discusses the practicability of manufacturing bioactive peptides, especially from food proteins.

A pH-sensitive, colorful, lanthanide-chelating paramagnetic NMR probe.

Paramagnetic lanthanides ions are broadly used in NMR spectroscopy. The effects of unpaired electrons on NMR spectral parameters provide a powerful tool for the characterization of macromolecular structures and dynamics. Here, a new lanthanide-chelating NMR probe, Caged Lanthanide NMR Probe-7 (CLaNP-7), is presented. It can be attached to protein surfaces via two disulfide bridges, yielding a probe that is rigid relative to the protein backbone. CLaNP-7 extends the application range of available probes. It has a yellow color, which is helpful for sample preparation. Its effects are comparable to those of CLaNP-5, but its charge is two units lower (+1) than that of CLaNP-5 (+3), reducing the change in surface potential after probe attachment. It also has a different magnetic susceptibility tensor, so by using both tags, two sets of structural restraints can be obtained per engineered cysteine pair. Moreover, it was found that the orientation of the magnetic susceptibility tensor is pH dependent (pK(a) ≈ 7) when a histidine residue is located in the neighborhood of the probe attachment site. The results show that the His imidazole group interacts with the CLaNP-7 tag. It is proposed that the histidine residue forms a hydrogen bond to a water/hydroxyl molecule that occupies the ninth coordination position on the lanthanide, thus breaking the two-fold symmetry of the CLaNP tag in a pH-dependent way.