Small multidrug resistance (SMR) transporters provide an ideal system to study the minimal requirements for active transport. EmrE is an E. coli SMR transporter that exports a broad class of polyaromatic cation substrates, thus conferring resistance to drug compounds matching this chemical description. However, a great deal of controversy has surrounded the topology of the EmrE homodimer. Here we show that asymmetric antiparallel EmrE exchanges between inward- and outward-facing states that are identical except that they have opposite orientation in the membrane. We quantitatively measure the global conformational exchange between these two states for substrate-bound EmrE in bicelles using solution NMR dynamics experiments. FRET reveals that the monomers within each dimer are antiparallel, and paramagnetic relaxation enhancement NMR experiments demonstrate differential water accessibility of the two monomers within each dimer. Our experiments reveal a “dynamic symmetry” that reconciles the asymmetric EmrE structure with the functional symmetry of residues in the active site.

Plasmodium parasites use specialized ligands which bind to red blood cell (RBC) receptors during invasion. Defining the mechanism of receptor recognition is essential for the design of interventions against malaria. Here, we present the structural basis for Duffy antigen (DARC) engagement by P. vivax Duffy binding protein (DBP). We used NMR to map the core region of the DARC ectodomain contacted by the receptor binding domain of DBP (DBP-RII) and solved two distinct crystal structures of DBP-RII bound to this core region of DARC. Isothermal titration calorimetry studies show these structures are part of a multi-step binding pathway, and individual point mutations of residues contacting DARC result in a complete loss of RBC binding by DBP-RII. Two DBP-RII molecules sandwich either one or two DARC ectodomains, creating distinct heterotrimeric and heterotetrameric architectures. The DARC N-terminus forms an amphipathic helix upon DBP-RII binding. The studies reveal a receptor binding pocket in DBP and critical contacts in DARC, reveal novel targets for intervention, and suggest that targeting the critical DARC binding sites will lead to potent disruption of RBC engagement as complex assembly is dependent on DARC binding. These results allow for models to examine inter-species infection barriers, Plasmodium immune evasion mechanisms, P. knowlesi receptor-ligand specificity, and mechanisms of naturally acquired P. vivax immunity. The step-wise binding model identifies a possible mechanism by which signaling pathways could be activated during invasion. It is anticipated that the structural basis of DBP host-cell engagement will enable development of rational therapeutics targeting this interaction.

Human ileal bile acid binding protein (I-BABP) is a member of the family of intracellular lipid-binding proteins and is thought to play a role in the enterohepatic circulation of bile salts. Our group has previously shown that human I-BABP binds two molecules of glycocholate (GCA) with low intrinsic affinity but an extraordinary high degree of positive cooperativity. Besides the strong positive cooperativity, human I-BABP exhibits a high degree of site selectivity in its interactions with GCA and glycochenodeoxycholate (GCDA), the two major bile salts in humans. In this study, on the basis of our first generation nuclear magnetic resonance (NMR) structure of the ternary complex of human I-BABP with GCA and GCDA, we introduced single-residue mutations at certain key positions in the binding pocket that might disrupt a hydrogen-bonding network, a likely way of energetic communication between the two sites. Macroscopic binding parameters were determined using isothermal titration calorimetry, and site selectivity was monitored by NMR spectroscopy of isotopically enriched bile salts. According to our results, cooperativity and site selectivity are not linked in human I-BABP. While cooperativity is governed by a subtle interplay of entropic and enthalpic contributions, site selectivity appears to be determined by more localized enthalpic effects. Possible communication pathways between the two binding sites are discussed.

The recognition between proteins and their native ligands is fundamental to biological function. In vivo, human ileal bile acid binding protein (I-BABP) encounters a range of bile salts that vary in the number and position of steroidal hydroxyl groups and the presence and type of side-chain conjugation. Therefore, it is necessary to understand how chemical variability in the ligand affects the energetic and structural aspects of its recognition. Here we report studies of the binding site selectivity of I-BABP for glycocholic (GCA) and glycochenodeoxycholic (GCDA) acids using isotope-enriched bile salts along with two-dimensional heteronuclear NMR methods. When I-BABP is presented with either GCA or GCDA alone, the ligands bind to both sites. However, when presented with an equimolar mixture of the two bile salts, GCDA binds exclusively to site 1 and GCA to site 2. This remarkable selectivity is governed by the presence or absence of a single hydroxyl group at the C-12 position of the steroid tetracycle. The basis for this site selectivity appears to be energetic rather then steric.

Wurtzite CdSe quantum belts (QBs) having Z-type ligation, such as {CdSe[Cd(oleate) 2 ] 0.19 } QBs, undergo a facile ligation exchange with AX salts (A = R 4 N, Na; X = OH, Cl, Br, NO 3 , OBz, OAc) to afford QBs having bound-ion-pair X-type ligation and empirical formulas CdSe[X] x [A] x . The exchange to AX ligation is accompanied by shifts of the quantum-belt absorption spectra by as large as 340 meV (for X = OH) relative to the spectrum of L-type {CdSe[noctylamine] 0.53 } QBs. AX ligation is also investigated using the Na + salts of D-and L-phenylalanine. These chiral X ligands induce inverse chiroptical effects in the circular dichroism spectra corresponding to the electronic transitions of the CdSe QBs, providing a strong evidence of the direct ligation of the X groups on the QB surfaces. AX ligation appears to consist of two populations on the QBs, one for which the AX ligands are persistently bound and another for which the AX ligands are readily removed by washing. These generate two ligation stoichiometries referred to as depleted and saturated ligation. The empirical formulas for the depleted ligation are in the approximate range of CdSe[X] 0.1−0.3 [A] 0.1−0.3 , whereas those for saturated ligation are in the approximate range of CdSe[X] 0.4−0.8 [A] 0.4−0.8 . These ranges are consistent with approximately one and two X − ligands per three-coordinate surface Cd atom. AX ligation is readily exchanged to either L-type primary amine or Z-type Cd(oleate) 2 ligation. However, AX salts do not displace L-type primary amine ligation under the conditions studied. All ligation exchanges are rapid and complete at room temperature, and with the exception of L-type to bound-ion-pair X-type ligation, fully reversible.