Probing amino acid side chains of the integral membrane protein PagP by solution NMR: Side chain immobilization facilitates association of secondary structures.

Solution NMR spectroscopy of large protein systems is hampered by rapid signal decay, so most multidimensional studies focus on long-lived 1H-13C magnetization in methyl groups and/or backbone amide 1H-15N magnetization in an otherwise perdeuterated environment. Herein we demonstrate that it is possible to biosynthetically incorporate additional 1H-12C groups that possess long-lived magnetization using cost-effective partially deuterated or unlabeled amino acid precursors added to Escherichia coli growth media. This approach is applied to the outer membrane enzyme PagP in membrane-mimetic dodecylphosphocholine micelles. We were able to obtain chemical shift assignments for a majority of side chain 1H positions in PagP using nuclear Overhauser enhancements (NOEs) to connect them to previously assigned backbone 1H-15N groups and newly assigned 1H-13C methyl groups. Side chain methyl-to-aromatic NOEs were particularly important for confirming that the amphipathic α-helix of PagP packs against its eight-stranded ß-barrel, as indicated by previous X-ray crystal structures. Interestingly, aromatic NOEs suggest that some aromatic residues in PagP that are buried in the membrane bilayer are highly mobile in the micellar environment, like Phe138 and Phe159. In contrast, Tyr87 in the middle of the bilayer is quite rigid, held in place by a hydrogen bonded network extending to the surface that resembles a classic catalytic triad: Tyr87-His67-Asp61. This hydrogen bonded arrangement of residues is not known to have any catalytic activity, but we postulate that its role is to immobilize Tyr87 to facilitate packing of the amphipathic α-helix against the ß-barrel.

Cost-effective selective deuteration of aromatic amino acid residues produces long-lived solution 1H NMR magnetization in proteins.

Solution NMR studies of large proteins are hampered by rapid signal decay due to short-range dipolar 1H-1H and 1H-13C interactions. These are attenuated by rapid rotation in methyl groups and by deuteration (2H), so selective 1H,13C-isotope labelling of methyl groups in otherwise perdeuterated proteins, combined with methyl transverse relaxation optimized spectroscopy (methyl-TROSY), is now standard for solution NMR of large protein systems > 25 kDa. For non-methyl positions, long-lived magnetization can be introduced as isolated 1H-12C groups. We have developed a cost-effective chemical synthesis for producing selectively deuterated phenylpyruvate and hydroxyphenylpyruvate. Feeding these amino acid precursors to E. coli in D2O, along with selectively deuterated anthranilate and unlabeled histidine, results in isolated and long-lived 1H magnetization in the aromatic rings of Phe (HD, HZ), Tyr (HD), Trp (HH2, HE3) and His (HD2 and HE1). We are additionally able to obtain stereoselective deuteration of Asp, Asn, and Lys amino acid residues using unlabeled glucose and fumarate as carbon sources and oxalate and malonate as metabolic inhibitors. Combining these approaches produces isolated 1H-12C groups in Phe, Tyr, Trp, His, Asp, Asn, and Lys in a perdeuterated background, which is compatible with standard 1H-13C labeling of methyl groups in Ala, Ile, Leu, Val, Thr, Met. We show that isotope labeling of Ala is improved using the transaminase inhibitor L-cycloserine, and labeling of Thr is improved through addition of Cys and Met, which are known inhibitors of homoserine dehydrogenase. We demonstrate the creation of long-lived 1H NMR signals in most amino acid residues using our model system, the WW domain of human Pin1, as well as the bacterial outer membrane protein PagP.

Proton TOCSY NMR relaxation rates quantitate protein side chain mobility in the Pin1 WW domain.

Protein side chain dynamics play a vital role in many biological processes, but differentiating mobile from rigid side chains remains a technical challenge in structural biology. Solution NMR spectroscopy is ideally suited for this but suffers from limited signal-to-noise, signal overlap, and a need for fractional 13C or 2H labeling. Here we introduce a simple strategy measuring initial 1H relaxation rates during a 1H TOCSY sequence like DIPSI-2, which can be appended to the beginning of any multi-dimensional NMR sequence that begins on 1H. The TOCSY RF field compels all 1H atoms to behave similarly under the influence of strong coupling and rotating frame cross-relaxation, so that differences in relaxation rates are due primarily to side chain mobility. We apply the scheme to a thermostable mutant Pin1 WW domain and demonstrate that the observed 1H relaxation rates correlate well with two independent NMR measures of side-chain dynamics, cross-correlated 13C relaxation rates in 13CßH2 methylene groups and maximum observable 3J couplings sensitive to the χ1 side chain dihedral angle (3JHα,Hß, 3JN,Hß, and 3JCO,Hß). The most restricted side chains belong to Trp26 and Asn40, which are closely packed to constitute the folding center of the WW domain. None of the other conserved aromatic residues is as immobile as the first tryptophan side chain of the WW domain. The proposed 1H relaxation methodology should make it relatively easy to measure side chain dynamics on uniformly 15N- or 13C-labeled proteins, so long as chemical shift assignments are obtainable.

Solution NMR spectroscopy of membrane proteins.

Integral membrane proteins (IMPs) perform unique and indispensable functions in the cell, making them attractive targets for fundamental research and drug discovery. Developments in protein production, isotope labeling, sample preparation, and pulse sequences have extended the utility of solution NMR spectroscopy for studying IMPs with multiple transmembrane segments. Here we review some recent applications of solution NMR for studying structure, dynamics, and interactions of polytopic IMPs, emphasizing strategies used to overcome common technical challenges.

The calcium sensitizer drug MCI-154 binds the structural C-terminal domain of cardiac troponin C.

The compound MCI-154 was previously shown to increase the calcium sensitivity of cardiac muscle contraction. Using solution NMR spectroscopy, we demonstrate that MCI-154 interacts with the calcium-sensing subunit of the cardiac troponin complex, cardiac troponin C (cTnC). Surprisingly, however, it binds only to the structural C-terminal domain of cTnC (cCTnC), and not to the regulatory N-terminal domain (cNTnC) that determines the calcium sensitivity of cardiac muscle. Physiologically, cTnC is always bound to cardiac troponin I (cTnI), so we examined its interaction with MCI-154 in the presence of two soluble constructs, cTnI1-77 and cTnI135-209, which contain all of the segments of cTnI known to interact with cTnC. Neither the cTnC-cTnI1-77 complex nor the cTnC-cTnI135-209 complex binds to MCI-154. Since residues 39-60 of cTnI are known to bind tightly to the cCTnC domain to form a structured core that is invariant throughout the cardiac cycle, we conclude that MCI-154 does not bind to cTnC when it is part of the intact cardiac troponin complex. Thus, MCI-154 likely exerts its calcium sensitizing effect by interacting with a target other than cardiac troponin.

Identification and Characterization of Novel Receptor-Interacting Serine/Threonine-Protein Kinase 2 Inhibitors Using Structural Similarity Analysis.

Receptor-interacting protein kinase 2 (RIP2 or RICK, herein referred to as RIPK2) is linked to the pathogen pathway that activates nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) and autophagic activation. Using molecular modeling (docking) and chemoinformatics analyses, we used the RIPK2/ponatinib crystal structure and searched in chemical databases for small molecules exerting binding interactions similar to those exerted by ponatinib. The identified RIPK2 inhibitors potently inhibited the proliferation of cancer cells by > 70% and also inhibited NFκB activity. More importantly, in vivo inhibition of intestinal and lung inflammation rodent models suggests effectiveness to resolve inflammation with low toxicity to the animals. Thus, our identified RIPK2 inhibitor may offer possible therapeutic control of inflammation in diseases such as inflammatory bowel disease, asthma, cystic fibrosis, primary sclerosing cholangitis, and pancreatitis.

Stereoselective Deuteration in Aspartate, Asparagine, Lysine, and Methionine Amino Acid Residues Using Fumarate as a Carbon Source for Escherichia coli in D2O.

Perdeuteration with selective 1H,13C-enrichment of methyl groups has enabled solution NMR studies of large (>30 kDa) protein systems. However, we propose that for all non-methyl positions, only magnetization originating from 1H-12C groups is sufficiently long-lived, and it can be transferred via through-space NOEs to slowly relaxing 1H-15N or 1H-13C methyl groups to achieve multidimensional solution NMR. We demonstrate stereoselective 1H,12C-labeling by adding relatively inexpensive unlabeled carbon sources to Escherichia coli growth media in D2O. Using our model system, a mutant WW domain from human Pin1, we compare deuteration patterns in 19 amino acids (all except cysteine). Protein grown using glucose as the sole carbon source had high levels of protonation in aromatic rings and the Hß positions of serine and tryptophan. In contrast, using our FROMP media (fumarate, rhamnose, oxalate, malonate, pyruvate), stereoselective protonation of Hß2 with deuteration at Hα and Hß3 was achieved in Asp, Asn, Lys, and Met residues. In solution NMR, stereospecific chemical shift assignments for Hß are typically obtained in conjunction with χ1 dihedral angle determinations using 3-bond J-coupling (3JN-Hß, 3JCO-Hß, 3JHα-Hß) experiments. However, due to motional averaging, the assumption of a pure rotameric state can yield incorrect χ1 dihedral angles with incorrect stereospecific assignments. This was the case for three residues in the Pin1 WW domain (Lys28, Met30, and Asn44). Thus, stereoselective 1H,12C-labeling will be useful not only for NMR studies of large protein systems, but also for determining side chain rotamers and dynamics in any protein system.