Honors & Awards
AAAS/Science Program for Excellence in Science, AAAS (2012–2014)
Doctor, Ludwig Maximilian Universitat Munchen (2010)
Brian Kobilka, Postdoctoral Faculty Sponsor
G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the β2-adrenergic receptor (β2AR) using (19)F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound β2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.
View details for DOI 10.1016/j.cell.2015.04.043
View details for Web of Science ID 000355152600017
View details for PubMedID 25981665
The Na(+)/proline symporter (PutP), like several other Na(+)-coupled symporters, belongs to the so-called LeuT-fold structural family, which features ten core transmembrane domains (cTMs) connected by extra- and intracellular loops. The role of these loops has been discussed in context with the gating function in the alternating access model of secondary active transport processes. Here we report the complete spin-labeling site scan of extracellular loop 4 (eL4) in PutP that reveals the presence of two α-helical segments, eL4a and eL4b. Among the eL4 residues that are directly implicated in the functional dynamics of the transporter, Phe314 in eL4b anchors the loop by means of hydrophobic contacts to cTM1 close to the ligand binding sites. We propose that ligand-induced conformational changes at the binding sites are transmitted via the anchoring residue to eL4 and through eL4 further to adjacent cTMs, leading to closure of the extracellular gate.
View details for DOI 10.1016/j.str.2014.03.011
View details for Web of Science ID 000335443700014
View details for PubMedID 24768113
Helicobacter pylori is cause of chronic gastritis, duodenal ulcer and gastric carcinoma in humans. L-proline is a preferred energy source of the microaerophilic bacterium. Previous analyses revealed that HpputP and HpputA, the genes that are predicted to play a central role in proline metabolism as they encode for the proline transporter and proline dehydrogenase, respectively, are essential for stomach colonization. Here, the molecular basis of proline transport in H. pylori by HpPutP was investigated experimentally for the first time. Measuring radiolabeled substrate transport in H. pylori and E. coli heterologously expressing HpputP as well as in proteoliposomes reconstituted with HpPutP, we demonstrate that the observed proline transport in H. pylori is mediated by HpPutP. HpPutP is specific and exhibits a high affinity for L-proline. Notably, L-proline transport is exclusively dependent on Na(+) as coupling ion, i.e., Na(+)/L-proline symport, reminiscent to the properties of PutP of E. coli even though H. pylori lives in a more acidic environment. Homology model-based structural comparisons and substitution analyses identified amino acids crucial for function. HpPutP-catalyzed proline uptake was efficiently inhibited by the known proline analogs 3,4-dehydro-D,L-proline and L-azetidine-2-carboxylic acid.
View details for DOI 10.1371/journal.pone.0083576
View details for Web of Science ID 000328737700062
View details for PubMedID 24358297
The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of ?-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of ?-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate ?-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of ?-arrestin-1. The structure of the ?-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in ?-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of ?-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on ?-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.
View details for DOI 10.1038/nature12120
View details for PubMedID 23604254
In the cyanobacterium Synechocystis sp PCC 6803, early steps in thylakoid membrane (TM) biogenesis are considered to take place in specialized membrane fractions resembling an interface between the plasma membrane (PM) and TM. This region (the PratA-defined membrane) is defined by the presence of the photosystem II (PSII) assembly factor PratA (for processing-associated TPR protein) and the precursor of the D1 protein (pD1). Here, we show that PratA is a Mn(2+) binding protein that contains a high affinity Mn(2+) binding site (K(d) = 73 ?M) and that PratA is required for efficient delivery of Mn(2+) to PSII in vivo, as Mn(2+) transport is retarded in pratA(-). Furthermore, ultrastructural analyses of pratA(-) depict changes in membrane organization in comparison to the wild type, especially a semicircle-shaped structure, which appears to connect PM and TM, is lacking in pratA(-). Immunogold labeling located PratA and pD1 to these distinct regions at the cell periphery. Thus, PratA is necessary for efficient delivery of Mn(2+) to PSII, leading to Mn(2+) preloading of PSII in the periplasm. We propose an extended model for the spatial organization of Mn(2+) transport to PSII, which is suggested to take place concomitantly with early steps of PSII assembly in biogenesis centers at the cell periphery.
View details for DOI 10.1105/tpc.111.093914
View details for Web of Science ID 000302131000021
View details for PubMedID 22319052
The Na?/L-proline transporter PutP is a member of the Na?/solute symporter family (TC 2A.21, SLC5), which contains several hundred proteins of pro- and eukaryotic origin. Within the family, the capability of L-proline uptake is restricted to proteins of prokaryotes. PutP contributes to the use of L-proline as a nutrient. In addition, the transporter may supply cells with compatible solute during adaptation to osmotic stress. Based on these and other functions, PutP is of significance for various bacteria-host interactions including the virulence of human pathogens. A homology model of Escherichia coli PutP was generated based on the crystal structure of the Vibrio parahaemolyticus Na+/galactose symporter. According to the model, PutP has a core structure of five plus five transmembrane domains forming an inverted repeat similar as originally revealed by the crystal structure of the Na+/leucine transporter LeuT. The homology model is experimentally verified by Cys cross-linking and site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy. The putative sites of Na? and L-proline binding are described, and a putative transport mechanism is discussed.
View details for DOI 10.2741/3955
View details for Web of Science ID 000300049600022
View details for PubMedID 22201772
Na(+)/solute symporters are essential membrane integrated proteins that couple the flow of Na(+) ions driven by electrochemical Na(+) gradients to the transport of solutes across biological membranes. Here, we used a combination of molecular modeling techniques and evolutionary conservation analysis to construct and validate a first model of the Na(+)/proline symporter PutP of Escherichia coli based on the crystal structure of the bacterial Na(+)/galactose symporter vSGLT. Ligand docking experiments were employed to gain information about residues involved in proline binding. The proposed model is consistent with the available experimental data and was further validated by amino acid substitutions and kinetic and protein chemical analyses. Combination of the results of molecular modeling and functional studies predicts the location and organization of the Na(+) and proline binding sites. Remarkably, as proposed computationally and discovered here experimentally, residues Y140, W244, and Y248 of transmembrane segments 4 and 7 are found to be particularly important for PutP function and suggested to participate in proline binding and/or gating.
View details for DOI 10.1016/j.jmb.2010.11.045
View details for Web of Science ID 000287281800006
View details for PubMedID 21130773
The backbone structure is determined by site-directed spin labeling, double electron electron resonance measurements of distances, and modeling in terms of a helix-loop-helix construct for a transmembrane domain that is supposed to line the translocation pathway in the 54.3 kDa Na(+)/proline symporter PutP of Escherichia coli. The conformational distribution of the spin labels is accounted for by a rotamer library. An ensemble of backbone models with a root mean-square deviation of less than 2 A is obtained. These models exhibit a pronounced kink near residue T341, which is involved in substrate binding. The kink may be associated with a hinge that allows the protein to open and close an inwardly oriented cavity.
View details for DOI 10.1016/j.bpj.2008.09.030
View details for Web of Science ID 000266376200023
View details for PubMedID 19134477
Selected residues of transmembrane domain (TM) IX were previously shown to play key roles in ligand binding and transport in members of the Na(+)/solute symporter family. Using the Na(+)/proline transporter PutP as a model, a complete Cys scanning mutagenesis of TM IX (positions 324 to 351) was performed here to further investigate the functional significance of the domain. G328, S332, Q345, and L346 were newly identified as important for Na(+)-coupled proline uptake. Placement of Cys at one of these positions altered K(m(pro)) (S332C and L346C, 3- and 21-fold decreased, respectively; Q345C, 38-fold increased), K(0.5(Na+)) (S332C, 13-fold decreased; Q345C, 19-fold increased), and/or V(max) [G328C, S332C, Q345C, and L346C, 3-, 22-, 2-, and 8-fold decreased compared to PutP(wild type), respectively]. Membrane-permeant N-ethylmaleimide inhibited proline uptake into cells containing PutP with Cys at distinct positions in the middle (T341C) and cytoplasmic half of TM IX (C344, L347C, V348C, and S351C) and had little or no effect on all other single Cys PutP variants. The inhibition pattern was in agreement with the pattern of labeling with fluorescein-5-maleimide. In addition, Cys placed into the cytoplasmic half of TM IX (C344, L347C, V348C, and S351C) was protected from fluorescein-5-maleimide labeling by proline while Na(+) alone had no effect. Membrane-impermeant methanethiosulfonate ethyltrimethylammonium modified Cys in the middle (A337C and T341C) and periplasmic half (L331C) but not in the cytoplasmic half of TM IX in intact cells. Furthermore, Cys at the latter positions was partially protected by Na(+) but not by proline. Based on these results, a model is discussed according to which residues of TM IX participate in the formation of ligand-sensitive, hydrophilic cavities in the protein that may reconstitute part of the Na(+) and/or proline translocation pathway of PutP.
View details for DOI 10.1016/j.jmb.2008.07.070
View details for Web of Science ID 000260024500006
View details for PubMedID 18692508
The Na+/solute symporter family comprises more than 400 members of pro- and eukaryotic origin. Using the Na+/proline transporter PutP of Escherichia coli as a model, the role of two conserved residues, Ser-340 and Thr-341, is investigated to obtain insights into the mechanism of transport catalyzed by members of this family. Substitution of these amino acids alters the transport kinetics of cells and proteoliposomes containing the PutP variants significantly. In particular, the apparent affinities for Na+ and Li+ are reduced by 2 orders of magnitude or more. Also proline binding is affected, albeit to a lesser extent than ion binding. Thereby, the presence of a hydroxyl group at position 341 is essential for high affinity ligand binding. Furthermore, Cys placed at position 340 or 341 reacts with sulfhydryl reagents of different polarity, indicating accessibility from the water phase. In addition, Cys cross-linking suggests proximity of the residues to other amino acids previously shown to be crucial for ligand binding. For these reasons it is suggested that Ser-340 and Thr-341 are located in a ligand translocation pathway. Furthermore, it is proposed that the side chain of Thr-341 directly participates in Na+ binding.
View details for DOI 10.1074/jbc.M706741200
View details for Web of Science ID 000253426500050
View details for PubMedID 18156179
Transient or partial formation of complexes between biomacromolecules is a general mechanism used to control cellular functions. Several of these complexes escape structure determination by crystallographic means. We developed a new approach for determining the structure of protein dimers in the native environment (e.g., in the membrane) with high resolution in cases where the structure of the two monomers is known. The approach is based on measurements of distance distributions between spin labels in the range between 2 and 6 nanometers by a pulsed electron paramagnetic resonance technique and explicit modeling of spin label conformations. By applying this method to the membrane protein homodimer of the Na(+)/H(+) antiporter NhaA of Escherichia coli, the structure of the presumably physiological dimer was determined. It reveals two points of contact between the two monomers, with one of them confirming results of earlier cross-linking experiments.
View details for DOI 10.1529/biophysj.107.109769
View details for Web of Science ID 000250577700032
View details for PubMedID 17704177
Amino acid transport is a ubiquitous phenomenon and serves a variety of functions in prokaryotes, including supply of carbon and nitrogen for catabolic and anabolic processes, pH homeostasis, osmoprotection, virulence, detoxification, signal transduction and generation of electrochemical ion gradients. Many of the participating proteins have eukaryotic relatives and are successfully used as model systems for exploration of transporter structure and function. Distribution, physiological roles, functional properties, and structure-function relationships of prokaryotic alpha-amino acid transporters are discussed.
View details for DOI 10.1007/s00232-006-0880-x
View details for Web of Science ID 000245834300007
View details for PubMedID 17417701
The pH dependence of the structure of the main Na(+)/H(+) antiporter NhaA of Escherichia coli is studied by continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) techniques on singly spin-labeled mutants. Residues 225 and 254 were selected for site-directed spin labeling, as previous work suggested that they are situated in domains undergoing pH-dependent structural changes. A well-defined distance of 4.4 nm between residues H225R1 in neighboring molecules is detected by a modulation in double electron-electron resonance data. This indicates that NhaA exists as a dimer, as previously suggested by a low-resolution electron density map and cross-linking experiments. The modulation depth decreases reversibly when pH is decreased from 8 to 5.8. A quantitative analysis suggests a dimerization equilibrium, which depends moderately on pH. Furthermore, the mobility and polarity of the environment of a spin label attached to residue 225 change only slightly with changing pH, while no other changes are detected by CW EPR. As antiporter activity of NhaA changes drastically in the studied pH range, residues 225 and 254 are probably located not in the sensor or ion translocation sites themselves but in domains that convey the signal from the pH sensor to the translocation site.
View details for DOI 10.1529/biophysj.105.062232
View details for Web of Science ID 000230822200054
View details for PubMedID 15894644