Mariusz Jaremko

Structure of the mitochondrial translocator protein in complex with a diagnostic ligand.

Translocation in Injury The translocator protein TSPO is essential for the import of cholesterol and porphyrins into mitochondria. TSPO expression increases in areas of brain injury and during neuroinflammation and, thus, has diagnostic and therapeutic implications. Jaremko et al. (p. 1363) used nuclear magnetic resonance spectroscopy to determine the high-resolution structure of the 18- kilodalton mammalian TSPO with the ligand PK11195, which stabilized the structure and resolved the conformation as a tight bundle of five helices. A ligand stabilizes a mammalian mitochondrial cholesterol transporter, allowing high-resolution structural analysis. The 18-kilodalton translocator protein TSPO is found in mitochondrial membranes and mediates the import of cholesterol and porphyrins into mitochondria. In line with the role of TSPO in mitochondrial function, TSPO ligands are used for a variety of diagnostic and therapeutic applications in animals and humans. We present the three-dimensional high-resolution structure of mammalian TSPO reconstituted in detergent micelles in complex with its high-affinity ligand PK11195. The TSPO-PK11195 structure is described by a tight bundle of five transmembrane α helices that form a hydrophobic pocket accepting PK11195. Ligand-induced stabilization of the structure of TSPO suggests a molecular mechanism for the stimulation of cholesterol transport into mitochondria.

Predictive Atomic Resolution Descriptions of Intrinsically Disordered hTau40 and α-Synuclein in Solution from NMR and Small Angle Scattering

The development of molecular descriptions of intrinsically disordered proteins (IDPs) is essential for elucidating conformational transitions that characterize common neurodegenerative disorders. We use nuclear magnetic resonance, small angle scattering, and molecular ensemble approaches to characterize the IDPs Tau and α-synuclein. Ensemble descriptions of IDPs are highly underdetermined due to the inherently large number of degrees of conformational freedom compared with available experimental measurements. Using extensive cross-validation we show that five different types of independent experimental parameters are predicted more accurately by selected ensembles than by statistical coil descriptions. The improvement increases in regions whose local sampling deviates from statistical coil, validating the derived conformational description. Using these approaches we identify enhanced polyproline II sampling in aggregation-nucleation sites, supporting suggestions that this region of conformational space is important for aggregation.

NMR structure of the human prion protein with the pathological Q212P mutation reveals unique structural features.

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrPC) conformer, denoted as infectious scrapie isoform or PrPSc. In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrPSc in specific brain regions leading to neuronal cell degeneration and death. Here, we describe the NMR solution structure of the truncated recombinant human PrP from residue 90 to 231 carrying the Q212P mutation, which is believed to cause Gerstmann-Sträussler-Scheinker (GSS) syndrome, a familial prion disease. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90–124) and a globular domain (residues 125–231). The substitution of a glutamine by a proline at the position 212 introduces novel structural differences in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the β2–α2 loop region. This structure might provide new insights into the early events of conformational transition of PrPC into PrPSc. Indeed, the spontaneous formation of prions in familial cases might be due to the disruptions of the hydrophobic core consisting of β2–α2 loop and α3 helix.

Folding of the Tau Protein on Microtubules.

Microtubules are regulated by microtubule-associated proteins. However, little is known about the structure of microtubule-associated proteins in complex with microtubules. Herein we show that the microtubule-associated protein Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules. While Tau is highly flexible in solution and adopts a β-sheet structure in amyloid fibrils, in complex with microtubules the conserved hexapeptides at the beginning of the Tau repeats two and three convert into a hairpin conformation. Thus, binding to microtubules stabilizes a unique conformation in Tau.

The immunosuppressive activity and solution structures of ubiquitin fragments

Recently, ubiquitin was suggested as a promising anti-inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin(50-59) sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well-defined conformation in methanol, its structure was distinct from the corresponding 50-59 fragment in the native ubiquitin molecule. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 423-431, 2009.

Cold denaturation of a protein dimer monitored at atomic resolution

Protein folding and unfolding are crucial for a range of biological phenomena and human diseases. Defining the structural properties of the involved transient species is therefore of prime interest. Using a combination of cold denaturation with NMR spectroscopy, we reveal detailed insight into the unfolding of the homodimeric repressor protein CylR2. Seven three-dimensional structures of CylR2 at temperatures from 25 °C to -16 °C reveal a progressive dissociation of the dimeric protein into a native-like monomeric intermediate followed by transition into a highly dynamic, partially folded state. The core of the partially folded state seems critical for biological function and misfolding.

Structure and Dynamics of the First Archaeal Parvulin Reveal a New Functionally Important Loop in Parvulin-type Prolyl Isomerases

Parvulins are a group of peptidyl-prolyl isomerases (PPIases) responsible for important biological processes in all kingdoms of life. The PinA protein from the psychrophilic archaeon Cenarchaeum symbiosum is a parvulin-like PPIase. Due to its striking similarity to the human parvulins Pin1 and Par14, PinA constitutes an interesting subject for structural and functional studies. Here, we present the first high resolution NMR structure of an archaeal parvulin, PinA, based on 1798 conformational restraints. Structure calculation yields an ensemble of 20 convergent low energy structures with a backbone r.m.s.d. value of 0.6 Å within the secondary structure elements. The overall fold of PinA comprises the β-α3-β-α-β2 fold typical for all parvulin structures known so far, but with helix III being a short 310-helix. A detailed comparison of this high resolution structure of the first archaeal PinA protein with bacterial and eukaryotic parvulin PPIase structures reveals an atypically large catalytic binding site. This feature provides an explanation for cold-adapted protein function. Moreover, the residues in and around 310-helix III exhibit strong intramolecular dynamics on a microsecond to millisecond timescale and display structural heterogeneity within the NMR ensemble. A putative peptide ligand was found for PinA by phage display and was used for 1H-15N-HSQC titrations. Again, the flexible region around 310-helix III as well as residues of the peptide binding pocket showed the strongest chemical shift perturbations upon peptide binding. The local flexibility of this region also was modulated by ligand binding. A glycine and two positively charged residues are conserved in most parvulin proteins in this flexible loop region, which may be of general functional importance for parvulin-type PPIases.

Electrospray ionization mass spectrometry as a method for studying the high-pressure denaturation of proteins.

High-pressure denaturation of proteins can provide important information concerning their folding and function. These studies require expensive and complicated equipment. In this paper, we present a new convenient method for studying high-pressure denaturation of proteins combining DHX (deuterium-hydrogen exchange) and electrospray ionization MS. Application of various values of pressure causes different degrees of protein unfolding resulting in molecules with a different number of protons available for exchange with deuterons. After decompression a protein refolds and a certain number of deuterons are trapped within the hydrophobic core of a refolded protein. Redissolving the deuterated protein in an aqueous buffer initiates the DHX of amides located on the protein surface only, which can be monitored under atmospheric pressure by MS. Depending on the degree of deuteration after high-pressure treatment, the DHX kinetics are different and indicate how many deuterons were trapped in the protein after refolding. The dependence of this number on pressure gives information on the denaturation state of a protein. The distribution of deuterium along the sequence of a high-pressure-denatured protein was studied the ECD (electron-capture-induced dissociation) on a Fourier-transform mass spectrometer, enabling the monitoring of high-pressure denaturation with single amino acid resolution.

Structural Integrity of the A147T Polymorph of Mammalian TSPO

Ligands of the transmembrane protein TSPO are used for imaging of brain inflammation, but a common polymorphism in TSPO complicates their application to humans. Here we determined the three‐dimensional structure and side‐chain dynamics of the A147T polymorph of mammalian TSPO in complex with the first‐generation ligand PK11195. We show that A147T TSPO is able to retain the same structural and dynamic profile as the wild‐type protein and thus binds PK11195 with comparable affinity. Our study is important for the design of more potent diagnostic and therapeutic ligands of TSPO.

Structure of the mammalian TSPO/PBR protein.

The 3D structure of the 18-kDa transmembrane (TM) protein TSPO (translocator protein)/PBR (peripheral benzodiazepine receptor), which contains a binding site for benzodiazepines, is important to better understand its function and regulation by endogenous and synthetic ligands. We have recently determined the structure of mammalian TSPO/PBR in complex with the diagnostic ligand PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; Jaremko et al. (2014) Science 343: , 1363-1366], providing for the first time atomic-level insight into the conformation of this protein, which is up-regulated in various pathological conditions including Alzheimers disease and Parkinsons disease. Here, we review the studies which have probed the structural properties of mammalian TSPO/PBR as well as the homologues bacterial tryptophan-rich sensory proteins (TspOs) over the years and provide detailed insight into the 3D structure of mouse TSPO (mTSPO)/PBR in complex with PK11195.