Solution structure and dynamics of the outer membrane enzyme PagP by NMR

Abstract

The bacterial outer membrane enzyme PagP transfers a palmitate chain from a phospholipid to lipid A. In a number of pathogenic Gram-negative bacteria, PagP confers resistance to certain cationic antimicrobial peptides produced during the host innate immune response. The global fold of Escherichia coli PagP was determined in both dodecylphosphocholine and n-octyl-␤-D-glucoside detergent micelles using solution NMR spectroscopy. PagP consists of an eight-stranded anti-parallel ␤-barrel preceded by an amphipathic ␣ helix. The ␤-barrel is well defined, whereas NMR relaxation measurements reveal considerable mobility in the loops connecting individual ␤-strands. Three amino acid residues critical for enzymatic activity localize to extracellular loops near the membrane interface, positioning them optimally to interact with the polar headgroups of lipid A. Hence, the active site of PagP is situated on the outer surface of the outer membrane. Because the phospholipids that donate palmitate in the enzymatic reaction are normally found only in the inner leaflet of the outer membrane, PagP activity may depend on the aberrant migration of phospholipids into the outer leaflet. This finding is consistent with an emerging paradigm for outer membrane enzymes in providing an adaptive response toward disturbances in the outer membrane.

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

“…In contrast, peaks from residues in the transmembrane helix (helix 2), and in helices 3 and 4, retained significant intensity. This result indicates that helices 3 and 4 of PLM are also tightly associated with the micelle, and is consistent with the solid-state 15 N NMR spectra of PLM in lipid bilayers, which indicate the presence of helical segments associated with the membrane surface. A similar profile is observed for Mat-8, although in this case protection from MnCl 2 extends over the four helices.…”

“…For uniformly 15 N-and 13 C-labeled proteins, ( 15 NH 4 ) 2 SO 4 and 13 C-labeled glucose were supplied to the M9 salts. 2 H-labeled proteins were obtained by growing the bacteria in M9 media dissolved in D 2 O, and selectively 15 Nlabeled samples were obtained by supplying individual 15 N-labeled amino acids to the media. SDS-PAGE was performed with the Tris-tricine system [38], and gels were stained with Coomassie blue G250.…”

“…Typically, it is very difficult to measure and assign a sufficient number of long-range NOE (nuclear Overhauser effect) distance constraints to determine protein folds, however, this limitation can be overcome by preparing weakly aligned micelle samples for the measurement of residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) that provide key orientation constraints for structure determination. Beta-barrels, the second major class of membrane proteins, have been more successfully studied in micelles, since their compact structures lend themselves to the applications of transverse relaxation optimized spectroscopy (TROSY) [12] pulse sequences for improved resolution and to NOE-based methods of structure determination [13][14][15].…”

“…The data suggest that the peaks in the PISEMA spectrum will have to be fitted with Pisa wheels of different tilts, in agreement with the solution NMR studies in micelles, showing that helices 1 and 2 have different orientations. According to the data in micelles, the peaks in the spectrum of 15 N-Leu labeled CHIF should account for Leu 17 and 19 in helix 1 preceding the transmembrane helix (helix 2), and Leu 22,27,28,35,37 in the transmembrane helix.…”

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

“…[1][2][3][4][5][6] However, the number of high-resolution polytopic membrane protein structures is limited. To date, there are six NMR solution structures of b-barrel membrane proteins [1][2][3][4][5][6] (compared to the 89 unique b-barrel structures deposited in the Protein Data Bank), all except for OprH have corresponding X-ray crystal structures.…”