The Paulino Lab is focused on elucidating the mechanism of action of membrane transporters (secondary- and primary-active) and channels on a molecular level. Although of high pharmacological relevance (> 60% of current drugs target membrane proteins), how their three-dimensional structure relates to their function and vice-versa is poorly understood and requires an interdisciplinary approach at the interface of biology, chemistry and physics. To address these questions, we employ cryo-electron microscopy and contrast our findings with comprehensive functional studies. Projects in our group are driven by the fundamental question of how membrane transporters work: how does the protein architecture translate into function; what is the exact mechanism of action during translocation of compounds from one side of the membrane to the other; how are they regulated; how is malfunction related to a disease? Over the past years, we have gained a particular interest in membrane transport proteins that fall out-of-the-box, challenging the conceptual boundaries present when classifying transport mechanisms into merely primary-active transporters, secondary-active transporters, or channels. It is becoming increasingly evident that in the course of evolution conserved protein architectures not only evolved from one another but can merge together to adapt to different environmental and cellular requirements.
Groovy Channels & Scramblases
The eukaryotic TMEM16 family is capable of a remarkable functional dichotomy, where members can work as Ca2+-activated Cl–-channels and/or lipid scramblases, which catalyse the bidirectional diffusion of lipids, i.e. phosphatidylserine, between both membrane leaflets. Our studies demonstrate that both functions are mediated by distinct conformations, which we termed the alternating pore/cavity mechanism. Whereas in the scramblase structure we observe a membrane-spanning and membrane-accessible cavity through which lipids can slide, the furrow is closed in the Cl–-channel structure to form a pore that allows the diffusion of ions through the membrane. We were able to identify structural elements that are directly linked to ligand-binding and regulate anion conduction via an electrostatic barrier and a potential gate in TMEM16 ion channels. By exploiting the advantages of cryo-EM we obtained insights into the dynamics present during lipid translocation in TMEM16 scramblases and how they might be regulated. As most data were obtained with the protein surrounded by lipids the data further demonstrate how the protein interacts with lipids and distorts the membrane, thereby decreasing the energy barrier for lipid movement. These studies provided a great step forward in understanding the mechanism of action and regulation of TMEM16 scramblases. Current work is focused in understanding in more depth the role of lipids and the effect of lipid composition on the proposed stepwise activation mechanism and to which extend this holds for all TMEM16 members. This work is done in collaboration with the group of Raimund Dutzler at the University of Zurich, Switzerland.
Anticancer drug target with an elevator mechanism
Other projects have, apart from a mechanistic question, also a social-economical application. This is demonstrated in our studies of the human neutral amino-acid transporter ASCT2 from the SLCA1 family that also comprises the human excitatory amino acid transporters EAAT1-5 and the prokaryotic GltPhand GltTk. ASCT2 is the main source of glutamine uptake in human cells, which is strongly linked to cancer cell growth, poor patient survival and a new hot-target in cancer therapy. We were able to solve the first structures of the human ASCT2 in a substrate-bound inward-occluded state and more recently a substrate-free inward-open state of ASCT2. The latter represents the first structure of any SLCA1 in an inward-open state, and answers a long-lasting key mechanistic question. It was known that these transporters work like an elevator, in which the substrate is translocated across the cell membrane by a large displacement of the transport domain, whereas a small movement of hairpin 2 (HP2) gates the extracellular access to the substrate-binding site. However, it has remained unclear how substrate binding and release is gated on the cytoplasmic side. Strikingly, our data show that the same structural element (HP2) serves as a gate in the inward-facing as in the outward-facing state, revealing that SLC1A transporters work as one-gate elevators instead of two-gate elevators as previously assumed. This observation is of great fundamental interest, but also has potential implications for drug design. A prominent consequences of the one-gate elevator mechanism is that large protein movements take place in the cell membrane during transport. We were able to identify several unassigned densities near the gate and surrounding the scaffold domain, which may represent potential allosteric binding sites and guide the design of lipidic-inhibitors for anticancer therapy. This work is done in collaboration with the group of Dirk Slotboom, one of the pioneers in SLCA1, and the bioinformatics and medicinal chemistry labs of Avner Schlessinger and Christof Grevwer.
A true chimera: a P-type ATPase hijacks a channel
P-type ATPases ubiquitously pump cations across biological membranes to maintain vital ion gradients. Among those, the chimeric K+ uptake system KdpFABC is unique. This 157kDa complex is expressed under stress conditions, when the external K+ concentration is too low for ubiquitous K+-transporters to maintain the internal potassium concentration. KdpFABC is composed of four subunits, whereby the KdpA subunit resembles a K+-channel and the KdpB subunit is classified as a P-type ATPase (primary-active transporter). While ATP hydrolysis is accomplished by the P-type ATPase subunit KdpB, K+ has been assumed to be transported by the channel-like subunit KdpA. A first crystal structure uncovered its overall topology, suggesting such a spatial separation of energizing and transporting units. By contrast, our KdpFABC structures led us to propose a so far unprecedented transport mechanism via an intersubunit tunnel through KdpA and KdpB. It units the alternating-access mechanism of actively pumping P-type ATPases with the high affinity and selectivity of K+-channels. This way, KdpFABC functions as a true chimeric complex, synergizing the best features of otherwise separately evolved transport mechanisms. More recently we gained further insights into the coupling mechanism, where a phenylalanine is directly linked to ion propagation and turnover by: (1) acting as a gatekeeper to prevent unspecific access to the PBS and CBS from the intersubunit tunnel, preventing an uncoupling of ATP hydrolysis from K+; (2) being involved in ion coordination and progression via its π-electron system; and (3) regulating the rate-limiting step of KdpFABC, the E1-P/E2-P transition. This work is done in collaboration with the group of Inga Hänelt at the Goethe University in Frankfurt, Germany.
Osmoregulatory ABC transporter
Recently, we were able to shed light into questions that had remained elusive for decades, on the osmoregulatory ABC transporter OpuA, which protects cells against hypertonicity. We identified a novel scaffolding domain and a structural element that acts as the ionic strength sensor, and show how binding of cyclic-di-AMP blocks transport. We propose a detailed mechanism of transport and a new striking dual regulatory mechanism that controls the intake of osmolytes in cells. It provides a framework for how different signals and regulation strategies are integrated in an essential membrane transport protein, whose activity would otherwise become lethal for the cell if not exquisitely controlled. Excitingly, these findings are among the first obtained for a full-length protein with bound cyclic-di-AMP – a newly discovered 2nd messenger in prokaryotes and eukaryotes. Our current work focusses on probing the conformational dynamics, identifying the effect of anionic lipids and thereby resolve in more detail the interplay between ionic strength and cyclic-di-AMP regulation. This work is done in collaboration with the lab of Bert Poolman.
Bilayer-mediated toppling mechanism
Gram-positive bacteria that lack biosynthesis pathways for micronutrients such as vitamins use a class of membrane proteins to acquire them from the environment. These membrane proteins are called energy-coupling factor (ECF) transporters and function in an unusual way. A membrane-embedded substrate-binding protein rotates within the membrane to bring a molecule from the outside to the inside of the cell. It was, however, not clear how this motion can occur within a bilayer environment. We use cryogenic electron microscopy at 200 kV to visualize an ECF transporter within a lipid bilayer. The transporter causes deformations to the surrounding lipid environment. These insights offer an explanation for how changes of the lipid environment enable such motion in the transport process. This work is done in collaboration with the group of Dirk Slotboom.
Sample thickness measurements for optimized data acquisition workflow
While it is known that sample thickness is a key player for cryo-EM data quality, this parameter is not taken into account during common data collections. We have set up an optimized data collection workflow for single particle cryo-electron microscopy (cryo-EM), which maximizes data collection efficiency. Via our approach, sample thickness can be determined before data acquisition, and this information used to identify optimal regions and restrict data collection to images with preserved high-resolution details. This quality over quantity approach, almost entirely eliminates the time- and storage-consuming collection of suboptimal images, which would be discarded at a later stage. This strategy is especially useful, if the speed of data collection is restricted by the microscope hardware and software, or if data transfer, data storage and computational power are a bottleneck. We were able to successfully implement it in commonly used data acquisition packages and make it compatible with the majority of commercially available electron microscopes via the open-source software SerialEM. Since several years, we have used this workflow in all projects derived from our group proving its versatility and efficiency experimentally.
There are so many more things to explore in transporters and channels. So much still to understand. Stay tuned to see on what else we are working…
Garibsingh RAA, Ndaru E, Garaeva AA, Shi Y, Zielewicz L, Zakrepine P, Bonomi M, Slotboom DJ, Paulino C‡, Grewer C‡, Schlessinger A‡, Rational design of ASCT2 inhibitors using an integrated experimental-computational approach, accepted in PNAS (2021);
Thangaratnarajah C, Rheinberger J, Paulino C‡, Slotboom DJ‡, Insights into the bilayer-mediated toppling mechanism of a folate-specific ECF transporter by cryo-EM, PNAS 24, 118(34) (2021);
Silberberg JM*, Corey RA*, Hielkema L*, Stock C, Stansfeld PJ‡, Paulino C‡ and Haenelt I‡, Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC. Nat Commun 12, 5098 (2021)
Rheinberger J, Oostergetel GT, Resch GP, Paulino C‡; Optimized cryo-EM data acquisition workflow by sample thickness determination. Acta Cryst. D77, 565-571 (2021); DOI:10.1107/S205979832100334X
Kalienkova V‡, Clerico Mosina V, Paulino C‡; The groovy TMEM16 family: molecular mechanisms of lipid scrambling and ion conduction. J Mol Biol: 166941 (2021);
Lam AKM‡, Rheinberger J, Paulino C‡, Dutzler R‡, Gating the pore of the calcium-activated chloride channel TMEM16A; Nat Comms 12: 719–13 (2021);
Hielkema L, Heuser T, Paulino C, Walter A, Cryo-Transmission Electron Microscopy. Book chapter: in IoP-IPEM ebook series in Physics and Engineering in Medicine and Biology. Imaging Modalities for Biological and Preclinical Research: A Compendium Volume 1, Pages I.5.b-1 to I.5.b-17 (2021); DOI 10.1088/978-0-7503-3059-6
Walter JD*, Hutter CAJ*, Garaeva AA*, Scherer M, Zimmermann I, Wyss M, Rheinberger J, Earp J, Egloff P, Sorgenfrei M, Hürlimann LM, Gonda I, Meier G, Remm S, Thavarasah S, Zimmer G, Slotboom DJ, Paulino C‡, Plattet P‡, Seeger MA‡; Highly potent bispecific sybodies neutralizing SARS-CoV-2. BioRxiv (2020)
Sikkema H, van den Noort M, Rheinberger J, Paulino C‡, Poolman B‡, Gating by ionic strength and safety check by cyclic-di-AMP in the ABC transporter OpuA. Science Advances 6, 47 eabd7697 (2020)
DOI:10.1126/sciadv.abd7697; press release
Garibsingh RA, Ndaru E, Garaeva AA, Bonomi M, Slotboom DJ, Paulino C‡,Grewer C‡, Schlessinger A‡; Structural basis for stereospecific inhibition of ASCT2 from rational design. BioRxiv (2020);
Kalienkova V., Alvadia C., Clerico Mosina V., Paulino C. Single-Particle Cryo-EM of Membrane Proteins in Lipid Nanodiscs. In: Expression, Purification, and Structural Biology of Membrane Proteins. Methods in Molecular Biology, vol 2127, (2020). Springer, Nature
Garaeva AA, Guskov A, Slotboom DJ‡ and Paulino C‡ A one-gate elevator mechanism for the human neutral amino acid transporter ASCT2. Nature Comm 10, 3427 (2019)
DOI:10.1038/s41467-019-11363-x; press release
Alvadia C*, Lim NK*, Clerico Mosina V*, Oostergetel GT, Dutzler R‡ and Paulino C‡, Cryo-EM structures and functional characterization of the lipid scramblase TMEM16F. eLife 8, 213 (2019)
DOI:10.7554/eLife.44365; press release
Kalienkova V, Clerico Mosina V, Bryner L, Oostergetel GT, Dutzler R‡ and Paulino C‡, Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM. eLife 8, 213 (2019)
DOI:10.7554/eLife.44364; press release
Stock C*, Hielkema L*, Tascon I*, Wunnicke D, Oostergetel GT, Askargorta M, Paulino C‡ and Haenelt I‡, Cryo-EM structures of KdpFABC reveal K+ transport mechanism via two inter-subunit half-channels. Nat Comm 9, 4971 (2018)
DOI:10.1038/s41467-018-07319-2; F1000 Prime Recommendation; press releases
Garaeva AA, Oostergetel GT, Gati C, Guskov A‡, Paulino C‡ and Slotboom DJ‡, Cryo-EM structure of the human neutral amino acid transporter ASCT2. Nat. Struc. Mol. Biol. 25, 515-521 (2018)
DOI:s41594-018-0076-y; press release
Paulino C, Neldner Y, Lam AKM, Kalienkova V, Brunner JD, Schenck S and Dutzler R, Structural basis for anion conduction in the calcium-activated chloride channel TMEM16A. eLife 6 e26232 (2017)
DOI: 10.7554/eLife.26232; Selected for an eLife Insights
Paulino C*, Wöhlert D*, Kapotova E, Yildiz Ö and Kühlbrandt W, Structure and transport mechanism of the archaeal Na+/H+ antiporter MjNhaP1. eLife 3 e03583 (2014)
Paulino C and Kühlbrandt W, pH and sodium-induced changes in a sodium/proton antiporter. eLife 3, e01412 (2014)
Calinescu O, Paulino C, Kühlbrandt W, Fendler K, Keeping it simple – transport mechanism and pH regulation in Na+/H+ exchangers. J Biol Chem 289, 13168-14176 (2014)
Goswami P*, Paulino C*, Hizlan D, Vonck J, Yildiz Ö, Kühlbrandt W, Structure of the archaeal Na+/H+ antiporter NhaP1 and functional role of transmembrane helix 1. EMBO J 30, 439-449 (2011)
‡corresponding author *co-first author