Requirements on Paramagnetic Relaxation Enhancement Data for Membrane Protein Structure Determination by NMR

SummaryNuclear magnetic resonance (NMR) structure calculations of the α-helical integral membrane proteins DsbB, GlpG, and halorhodopsin show that distance restraints from paramagnetic relaxation enhancement (PRE) can provide sufficient structural information to determine their structure with an accuracy of about 1.5 Å in the absence of other long-range conformational restraints. Our systematic study with simulated NMR data shows that about one spin label per transmembrane helix is necessary for obtaining enough PRE distance restraints to exclude wrong topologies, such as pseudo mirror images, if only limited other NMR restraints are available. Consequently, an experimentally realistic amount of PRE data enables α-helical membrane protein structure determinations that would not be feasible with the very limited amount of conventional NOESY data normally available for these systems. These findings are in line with our recent first de novo NMR structure determination of a heptahelical integral membrane protein, proteorhodopsin, that relied extensively on PRE data

Strategien zur strukturellen Charakterisierung alpha-helikaler Membranproteine mittels zellfreier Expression und NMR Spektroskopie : eine Anwendung auf Proteorhodopsin

Membrane proteins (MPs) constitute about 30% of the genome and are essential in many cellular processes. In particular structural characterisation of MPs is challenged by their hydrophobic nature resulting in expression difficulties and structural instability upon extraction from the membrane. Despite these challenges, progress in sample preparation and the techniques to solve MP structures has led to 281 unique MP structures as of January 2011. Through the combination of a cell-free expression system and selective labelling strategies, this thesis aimed to advance the structure determination of α-helical MPs by NMR spectroscopy and resulted in the structure determination of a seven-ransmembrane-helix protein. Results were obtained for the 5-lipoxygenase-activating protein (FLAP) and proteorhodopsin (PR). The detergent-based cell-free expression mode proved most efficient for production of both targets, but optimisation of FLAP and PR followed different routes. The presence of a retinal cofactor in PR greatly facilitated the search for an appropriate hydrophobic environment. For structural studies, NMR spectra of FLAP indicated favourable properties of the lysolipid LPPG. In contrast, PR was stable and homogenous in the short-chain lipid diC7PC. As NMR spectra of α-helical MPs are generally characterised by broad lines and signal overlap, selective labelling strategies were essential in the assignment process of both targets. For the backbone assignment of FLAP the transmembrane segment-enhanced (TMS) labelling was developed, employing the six amino acids AFGILV. These residues cluster predominantly in transmembrane helices and form long stretches allowing a large extent of backbone assignment. Besides that, the combinatorial labelling enables identification of unique pairs in the sequence based on a mixture of 15N and 1-13C-labelled amino acids. To find the optimal labelling pattern for a given primary structure, the UPLABEL algorithm has been made available and successfully applied in the backbone assignment of PR. Both selective labelling approaches greatly benefitted from the use of a cell-free expression system to reduce isotope scrambling. Additionally, the de novo structure of PR was determined with an average backbone rmsd of 1.2 Å based on TALOS-derived backbone torsion angles, intrahelical hydrogen bond restraints and distance restraints from the NOE and paramagnetic relaxation enhancement (PRE). A major bottleneck in the NMR structure determination of MPs concerns the number of long-range distances which are often limited. In PR, side chain assignment was enabled by stereo-array isotope labelling as well as selective labelling which provided 33 long-range NOEs. These NOEs stabilised the symmetry of the seven helix bundle. With a total number of 1031, the majority of long-range distances were derived from PREs. The structure of PR reveals differences to its homologues such as the absence of an anti-parallel β-sheet between helices B and C and allows conclusions towards the mechanism of colour tuning.Ziel dieser Arbeit ist, Strategien zur Strukturaufklärung α-helikaler Membranproteine mittels NMR Spektroskopie weiter zu entwickeln und so das Spektrum möglicher Zielproteine zu erweitern. Auf Basis eines zellfreien Expressionssystems wurden Methoden zur selektiven Isotopenmarkierung erarbeitet und die drei-dimensionale Struktur einen Sieben-Transmembran-Helix Proteins bestimmt. Membranproteine spielen eine essentielle Rolle in vielen zellulären Prozessen wie dem Membrantransport, der Signaltransduktion oder in Zell-Zell Kontakten. Sie sind daher wichtige Zielproteine für die Entwicklung pharmazeutischer Wirkstoffe. Um Wirkstoffe gezielter einzusetzen und sie zu verbessern, ist ein tiefgreifendes Verständnis dieser Proteine erforderlich. Die funktionelle und strukturelle Erforschung von Membranproteinen ist jedoch durch deren hydrophoben Eigenschaften erschwert. Hierbei kommt es zu Expressionsschwierigkeiten und einer erhöhten Instabilität der Proteine außerhalb der Zellmembran. Dies betrifft insbesondere Proteine mit überwiegend α-helikaler Sekundärstruktur, welche die Mehrheit der Membranproteine darstellen. Vor allem für die Strukturanalyse werden große Mengen an stabilem und homogenem Protein benötigt. Nichts desto trotz gelang es in den vergangenen Jahren einige Strukturen helikaler Membranproteine einschließlich G-Protein gekoppelter Rezeptoren aufzuklären. Dieser Erfolg lässt sich zurückführen auf Verbesserungen bei der Probenherstellung sowie auf technische Forschritte der Methoden zur Strukturaufklärung. Nur einige wenige Membranproteine liegen in hohen zellulären Konzentrationen vor, während der Großteil der Zielproteine hingegen heterolog exprimiert werden muss. Bakterielle Expression in E. coli bietet hierfür ein breites Methodenspektrum. Höhere Expressionssysteme sind aber vor allem bei eukaryotischen Proteinen vielversprechend. So ermöglichte beispielsweise die Insektenzell-Expression in Sf9 Zellen die Strukturaufklärung des β2-Adrenergen Rezeptors (1). Eine alternative Methode zur Herstellung ausreichender Mengen Membranprotein ist die zellfreie Expression. Dieses offene System ermöglicht eine direkte Kontrolle des Reaktionsprotokolls und eröffnet somit verschiedene Modi zur Expression von Membranproteinen entweder in Anwesenheit von Detergenz oder Lipid oder gänzlich ohne hydrophobe Umgebung. Wird keine hydrophobe Umgebung zugesetzt, präzipitiert das Zielprotein. Dieses Präzipitat unterscheidet sich jedoch von den Einschlusskörpern in E. coli und lässt sich in relativ milden Detergenzien resolubilisieren. Die Anwesenheit von Detergenz in der zellfreien Reaktion erlaubt die lösliche Expression des Proteins und als dritte Option besteht die Möglichkeit eines Lipid-basierten Expressionsmodus. Auf diese Weise ergeben sich vielfältige Wege zur Optimierung des Zielproteins. Einen großen Vorteil bietet das zellfreie Expressionssystem hinsichtlich der Verwendung markierter oder unnatürlicher Aminosäuren. Dies gilt vor allem für die NMR Spektroskopie, denn isotopenmarkierte Aminosäuren werden nur bedingt in andere Aminosäuren konvertiert. .

Perspectives of Solution NMR Spectroscopy for Structural and Functional Studies of Integral Membrane Proteins

This article discusses future perspectives of solution NMR spectroscopy to study structures and functions of integral membrane proteins at atomic resolution, based on a review of recent progress in this area. Several selected examples of structure determinations, as well as functional studies of integral membrane proteins are highlighted. We expect NMR spectroscopy to make future key scientific contributions to understanding membrane protein function, in particular for large membrane protein systems with known three-dimensional structure. Such situations can benefit from the fact that functional NMR studies have substantially less limitations by molecular size than a full de novo structure determination. Therefore, the general potential for NMR spectroscopy to solve biologic key questions associated with integral membrane proteins is very promising

Kinase Regulation in Mycobacterium tuberculosis: Variations on a Theme

In this issue of Structure, Lisa et al. (2015) examine how the PknG protein kinase of M. tuberculosis efficiently binds and phosphorylates substrates. The work highlights interesting parallels between PknG and eukaryotic protein kinases

Improved accuracy in measuring one-bond and two-bond 15N,13Ca coupling constants in proteins by double-inphase/antiphase (DIPAP) spectroscopy

An extension to HN(CO-a/b-N,Ca-J)-TROSY (Permi and Annila in J Biomol NMR 16:221–227, 2000) is proposed that permits the simultaneous determination of the four coupling constants 1JN'(i)Ca(i), 2JHN(i)Ca(i), 2JCa(i-1)N'(i), and 3JCa(i-1)HN(i) in 15N,13C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the 2JCaN' coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase and antiphase splittings possible with respect to both 2JCaN' and 1JN'Ca (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous 2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision. Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are exemplified using proteins of various size

Rapid Screen for Tyrosine Kinase Inhibitor Resistance Mutations and Substrate Specificity

We present a rapid and high-throughput yeast and flow cytometry based method for predicting kinase inhibitor resistance mutations and determining kinase peptide substrate specificity. Despite the widespread success of targeted kinase inhibitors as cancer therapeutics, resistance mutations arising within the kinase domain of an oncogenic target present a major impediment to sustained treatment efficacy. Our method, which is based on the previously reported YESS system, recapitulated all validated BCR-ABL1 mutations leading to clinical resistance to the second-generation inhibitor dasatinib, in addition to identifying numerous other mutations which have been previously observed in patients, but not yet validated as drivers of resistance. Further, we were able to demonstrate that the newer inhibitor ponatinib is effective against the majority of known single resistance mutations, but ineffective at inhibiting many compound mutants. These results are consistent with preliminary clinical and in vitro reports, indicating that mutations providing resistance to ponatinib are significantly less common; therefore, predicting ponatinib will be less susceptible to clinical resistance relative to dasatinib. Using the same yeast-based method, but with random substrate libraries, we were able to identify consensus peptide substrate preferences for the SRC and LYN kinases. ABL1 lacked an obvious consensus sequence, so a machine learning algorithm utilizing amino acid covariances was developed which accurately predicts ABL1 kinase peptide substrates

Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms