Andi Mainz

Large Protein Complexes with Extreme Rotational Correlation Times Investigated in Solution by Magic-Angle-Spinning NMR Spectroscopy

We show that large protein complexes can be investigated in solution using magic-angle-spinning (MAS) NMR spectroscopy without the need for sample crystallization or precipitation. In order to efficiently average anisotropic interactions with MAS, the rotational diffusion of the molecule has to be suppressed. This can be readily achieved by lowering the sample temperature and by adding glycerol to the protein solution. The approach is demonstrated using the human small heat shock protein (sHSP) alphaB-Crystallin, which forms oligomeric assemblies of approximately 600 kDa. We suggest this scheme as an approach for overcoming size limitations imposed by overall tumbling in solution-state NMR investigations of large protein complexes.

The gyrase inhibitor albicidin consists of p -aminobenzoic acids and cyanoalanine

Albicidin is a potent DNA gyrase inhibitor produced by the sugarcane pathogenic bacterium Xanthomonas albilineans. Here we report the elucidation of the hitherto unknown structure of albicidin, revealing a unique polyaromatic oligopeptide mainly composed of p-aminobenzoic acids. In vitro studies provide further insights into the biosynthetic machinery of albicidin. These findings will enable structural investigations on the inhibition mechanism of albicidin and its assessment as a highly effective antibacterial drug.

Nonribosomal Peptide Synthesis-Principles and Prospects

Nonribosomal peptide synthetases (NRPSs) are large multienzyme machineries that assemble numerous peptides with large structural and functional diversity. These peptides include more than 20 marketed drugs, such as antibacterials (penicillin, vancomycin), antitumor compounds (bleomycin), and immunosuppressants (cyclosporine). Over the past few decades biochemical and structural biology studies have gained mechanistic insights into the highly complex assembly line of nonribosomal peptides. This Review provides state-of-the-art knowledge on the underlying mechanisms of NRPSs and the variety of their products along with detailed analysis of the challenges for future reprogrammed biosynthesis. Such a reprogramming of NRPSs would immediately spur chances to generate analogues of existing drugs or new compound libraries of otherwise nearly inaccessible compound structures.

The chaperone αB-crystallin uses different interfaces to capture an amorphous and an amyloid client

Small heat-shock proteins, including αB-crystallin (αB), play an important part in protein homeostasis, because their ATP-independent chaperone activity inhibits uncontrolled protein aggregation. Mechanistic details of human αB, particularly in its client-bound state, have been elusive so far, owing to the high molecular weight and the heterogeneity of these complexes. Here we provide structural insights into this highly dynamic assembly and show, by using state-of-the-art NMR spectroscopy, that the αB complex is assembled from asymmetric building blocks. Interaction studies demonstrated that the fibril-forming Alzheimers disease Aβ1–40 peptide preferentially binds to a hydrophobic edge of the central β-sandwich of αB. In contrast, the amorphously aggregating client lysozyme is captured by the partially disordered N-terminal domain of αB. We suggest that αB uses its inherent structural plasticity to expose distinct binding interfaces and thus interact with a wide range of structurally variable clients.

Structural and Mechanistic Implications of Metal Binding in the Small Heat-shock Protein αB-crystallin

Background: αB-crystallin is an ATP-independent chaperone that prevents irreversible protein aggregation. Results: Cu(II) binds to the core domain of αB-crystallin, induces increased dynamics at the dimer interface, and thus modulates the anti-aggregation properties of the chaperone. Conclusion: The small heat-shock protein αB-crystallin is a metal-regulated chaperone. Significance: The results open new perspectives in the field of protein homeostasis and oxidative stress resistance. The human small heat-shock protein αB-crystallin (αB) rescues misfolded proteins from irreversible aggregation during cellular stress. Binding of Cu(II) was shown to modulate the oligomeric architecture and the chaperone activity of αB. However, the mechanistic basis of this stimulation is so far not understood. We provide here first structural insights into this Cu(II)-mediated modulation of chaperone function using NMR spectroscopy and other biophysical approaches. We show that the α-crystallin domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion with picomolar binding affinity. Putative Cu(II) ligands are His83, His104, His111, and Asp109 at the dimer interface. These loop residues are conserved among different metazoans, but also for human αA-crystallin, HSP20, and HSP27. The involvement of Asp109 has direct implications for dimer stability, because this residue forms a salt bridge with the disease-related Arg120 of the neighboring monomer. Furthermore, we observe structural reorganization of strands β2-β3 triggered by Cu(II) binding. This N-terminal region is known to mediate both the intermolecular arrangement in αB oligomers and the binding of client proteins. In the presence of Cu(II), the size and the heterogeneity of αB multimers are increased. At the same time, Cu(II) increases the chaperone activity of αB toward the lens-specific protein βL-crystallin. We therefore suggest that Cu(II) binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher order assemblies of αB.

Paenilamicin: Structure and Biosynthesis of a Hybrid Nonribosomal Peptide/Polyketide Antibiotic from the Bee Pathogen Paenibacillus larvae

The spore-forming bacterium Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a fatal disease of honey bees that occurs worldwide. Previously, we identified a complex hybrid nonribosomal peptide/polyketide synthesis (NRPS/PKS) gene cluster in the genome of P. larvae. Herein, we present the isolation and structure elucidation of the antibacterial and antifungal products of this gene cluster, termed paenilamicins. The unique structures of the paenilamicins give deep insight into the underlying complex hybrid NRPS/PKS biosynthetic machinery. Bee larval co-infection assays reveal that the paenilamicins are employed by P. larvae in fighting ecological niche competitors and are not directly involved in killing the bee larvae. Their antibacterial and antifungal activities qualify the paenilamicins as attractive candidates for drug development.

Dynamics in the solid-state: perspectives for the investigation of amyloid aggregates, membrane proteins and soluble protein complexes

Aggregates formed by amyloidogenic peptides and proteins and reconstituted membrane protein preparations differ significantly in terms of the spectral quality that they display in solid-state NMR experiments. Structural heterogeneity and dynamics can both in principle account for that observation. This perspectives article aims to point out challenges and limitations, but also potential opportunities in the investigation of these systems.

Experimental determination of microsecond reorientation correlation times in protein solutions.

Reorientation correlation times in protein solutions are key determinants for feasibility and quality of NMR experiments. Yet, their accurate estimate is not easy, especially in the case of very large proteins. We show that nuclear magnetic relaxation dispersion (NMRD) can accurately determine reorientation times up to the microsecond range. A theoretical description for the analysis of the NMRD profiles is provided, and the protein reorientation time is shown to be provided by the longest correlation time among those needed to reproduce the experimental profile. Measurements are performed using samples of the archaeal proteasome double ring α7α7 and of αB-Crystallin in glycerol solutions.

The mechanism of denaturation and the unfolded state of the α-helical membrane-associated protein Mistic.

In vitro protein-folding studies using chemical denaturants such as urea are indispensible in elucidating the forces and mechanisms determining the stability, structure, and dynamics of water-soluble proteins. By contrast, α-helical membrane-associated proteins largely evade such approaches because they are resilient to extensive unfolding. We have used optical and NMR spectroscopy to provide an atomistic-level dissection of the effects of urea on the structure and dynamics of the α-helical membrane-associated protein Mistic as well as its interactions with detergent and solvent molecules. In the presence of the zwitterionic detergent lauryl dimethylamine oxide, increasing concentrations of urea result in a complex sequence of conformational changes that go beyond simple two-state unfolding. Exploiting this finding, we report the first high-resolution structural models of the urea denaturation process of an α-helical membrane-associated protein and its completely unfolded state, which contains almost no regular secondary structure but nevertheless retains a topology close to that of the folded state.

The Albicidin Resistance Factor AlbD Is a Serine Endopeptidase That Hydrolyzes Unusual Oligoaromatic-Type Peptides.

The para-aminobenzoic acid-containing peptide albicidin is a pathogenicity factor synthesized by Xanthomonas albilineans in infections of sugar cane. Albicidin is a nanomolar inhibitor of the bacterial DNA gyrase with a strong activity against various Gram-negative bacteria. The bacterium Pantoea dispersa expresses the hydrolase AlbD, conferring natural resistance against albicidin. We show that AlbD is a novel type of endopeptidase that catalyzes the cleavage of albicidin at a peptide backbone amide bond, thus abolishing its antimicrobial activity. Additionally, we determined the minimal cleavage motif of AlbD with substrates derived by chemical synthesis. Our results clearly identify AlbD as a unique endopeptidase that is the first member of a new subfamily of peptidases. Our findings provide the molecular basis for a natural detoxification mechanism, potentially rendering a new tool in biological chemistry approaches.