Peter M. Hwang

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

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-β-d-glucoside detergent micelles using solution NMR spectroscopy. PagP consists of an eight-stranded anti-parallel β-barrel preceded by an amphipathic α helix. The β-barrel is well defined, whereas NMR relaxation measurements reveal considerable mobility in the loops connecting individual β-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.

Lipopeptide detergents designed for the structural study of membrane proteins

The structural study of membrane proteins requires detergents that can effectively mimic lipid bilayers, and the choice of detergent is often a compromise between detergents that promote protein stability and detergents that form small micelles. We describe lipopeptide detergents (LPDs), a new class of amphiphile consisting of a peptide scaffold that supports two alkyl chains, one anchored to each end of an α-helix. The goal was to design a molecule that could self-assemble into a cylindrical micelle with a rigid outer hydrophilic shell surrounding an inner lipidic core. Consistent with this design, LPDs self-assemble into small micelles, can disperse phospholipid membranes, and are gentle, nondenaturing detergents that preserve the structure of the membrane proteins in solution for extended periods of time. The LPD design allows for a membrane-like packing of the alkyl chains in the core of the molecular assemblies, possibly explaining their superior properties relative to traditional detergents in stabilizing membrane protein structures.

A hydrocarbon ruler measures palmitate in the enzymatic acylation of endotoxin

The ability of enzymes to distinguish between fatty acyl groups can involve molecular measuring devices termed hydrocarbon rulers, but the molecular basis for acyl‐chain recognition in any membrane‐bound enzyme remains to be defined. PagP is an outer membrane acyltransferase that helps pathogenic bacteria to evade the host immune response by transferring a palmitate chain from a phospholipid to lipid A (endotoxin). PagP can distinguish lipid acyl chains that differ by a single methylene unit, indicating that the enzyme possesses a remarkably precise hydrocarbon ruler. We present the 1.9 Å crystal structure of PagP, an eight‐stranded β‐barrel with an unexpected interior hydrophobic pocket that is occupied by a single detergent molecule. The buried detergent is oriented normal to the presumed plane of the membrane, whereas the PagP β‐barrel axis is tilted by approximately 25°. Acyl group specificity is modulated by mutation of Gly88 lining the bottom of the hydrophobic pocket, thus confirming the hydrocarbon ruler mechanism for palmitate recognition. A striking structural similarity between PagP and the lipocalins suggests an evolutionary link between these proteins.

Solution structures of the cytoplasmic tail complex from platelet integrin alpha IIb- and beta 3-subunits.

Integrin adhesion receptors constitute a cell-signaling system whereby interactions in the small cytoplasmic domains of the heterodimeric α- and β-subunits provoke major functional alterations in the large extracellular domains. With two-dimensional NMR spectroscopy, we examined two synthetic peptides [αIIb(987MWKVGFFKRNR) and β3(716KLLITIHDRKEFAKFEEERARAKWD)] encompassing the membrane-proximal regions of the cytoplasmic domain motifs from the platelet integrin complex αΙΙbβ3. These membrane-proximal regions contain two conserved motifs, represented by 989KVGFFKR in the αIIb-subunit, and 716KLLITIHDR in the β3-subunit. The dimer interaction consists of two adjacent helices with residues V990 and F993 of the αΙΙb-subunit heavily implicated in the dimer interfacial region, as is I719 of β3. These residues are situated within the conserved motifs of their respective proteins. Further structural analysis of this unique peptide heterodimer suggests that two distinct conformers are present. The major structural difference between the two conformers is a bend in the β3-peptide between D723 and A728, whereas the helical character in the other regions remains intact. Earlier mutational analysis has shown that a salt bridge between the side chains of αΙΙb(R955) and β3(D723) is formed. When this ion pair was modeled into both conformers, increased nuclear Overhauser effect violations suggested that the more bent structure was less able to accommodate this interaction. These results provide a molecular level rationalization for previously reported biochemical studies, as well as a basis for an atomic level understanding of the intermolecular interactions that regulate integrin activity.

A ‘three-pronged’ binding mechanism for the SAP/SH2D1A SH2 domain: structural basis and relevance to the XLP syndrome

The SH2 domain protein SAP/SH2D1A, encoded by the X‐linked lymphoproliferative (XLP) syndrome gene, associates with the hematopoietic cell surface receptor SLAM in a phosphorylation‐independent manner. By screening a repertoire of synthetic peptides, the specificity of SAP/SH2D1A has been mapped and a consensus sequence motif for binding identified, T/S‐x‐x‐x‐x‐V/I, where x represents any amino acid. Remarkably, this motif contains neither a Tyr nor a pTyr residue, a hallmark of conventional SH2 domain–ligand interactions. The structures of the protein, determined by NMR, in complex with two distinct peptides provide direct evidence in support of a ‘three‐pronged’ binding mechanism for the SAP/SH2D1A SH2 domain in contrast to the ‘two‐pronged’ binding for conventional SH2 domains. Differences in the structures of the two complexes suggest considerable flexibility in the SH2 domain, as further confirmed and characterized by hydrogen exchange studies. The structures also explain binding defects observed in disease‐causing SAP/SH2D1A mutants and suggest that phosphorylation‐independent interactions mediated by SAP/SH2D1A likely play an important role in the pathogenesis of XLP.

Domain orientation in β-cyclodextrin-loaded maltose binding protein: Diffusion anisotropy measurements confirm the results of a dipolar coupling study

Maltose binding protein (MBP) is a 370-residue two-domain molecule involved in bacterial chemotaxis and sugar uptake. Rotational diffusion tensors were calculated for a complex between MBP and β-cyclodextrin using backbone 15N T1 and T1ρ relaxation times and steady state 1H-15N NOE values. The tensors obtained for each of the two domains in the protein were subsequently used to determine the relative domain orientation in the molecule. The average domain orientation determined using this approach agrees well with results from dipolar coupling data, but differs significantly from the domain orientation deduced from X-ray studies of the complex.

Solution Structure and Dynamics of Integral Membrane Proteins by NMR: A Case Study Involving the Enzyme PagP

Solution NMR spectroscopy is rapidly becoming an important technique for the study of membrane protein structure and dynamics. NMR experiments on large perdeuterated proteins typically exploit the favorable relaxation properties of backbone amide (15)N-(1)H groups to obtain sequence-specific chemical shift assignments, structural restraints, and a wide range of dynamics information. These methods have proven successful in the study of the outer membrane enzyme, PagP, not only for obtaining the global fold of the protein but also for characterizing in detail the conformational fluctuations that are critical to its activity. NMR methods can also be extended to take advantage of slowly relaxing methyl groups, providing additional probes of structure and dynamics at side chain positions. The current work on PagP demonstrates how solution NMR can provide a unique atomic resolution description of the dynamic processes that are key to the function of many membrane protein systems.