Scientists in the Robia lab work on a variety of projects focused on protein structure-function relationships and molecular physiology. Major biological themes are summarized below, or check out our complete list of publications.

Inhibitory and Stimulatory Micropeptides Preferentially Bind to Different Conformations of the Cardiac Calcium Pump

The ATP-dependent ion pump sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. We hypothesized that the functions of these peptides may relate to their reciprocal preferences for SERCA binding; SERCA binds PLB more avidly at low cytoplasmic [Ca2+] but binds DWORF better when [Ca2+] is high. In the present study, we demonstrated this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca2+ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca2+]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca2+ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca2+ elevations. A computational model showed that DWORF exaggerates changes in PLB-SERCA binding during the cardiac cycle. These results suggest a mechanistic basis for inhibitory versus stimulatory micropeptide function, as well as a new role for DWORF as a modulator of dynamic oscillations of PLB-SERCA regulatory interactions.

Fluorescence Lifetime Imaging Microscopy Reveals Sodium Pump Dimers in Live Cells

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)2.

FXYD Proteins and Sodium Pump Regulatory Mechanisms

The sodium/potassium-ATPase (NKA) is the enzyme that establishes gradients of sodium and potassium across the plasma membrane. NKA activity is tightly regulated for different physiological contexts through interactions with single-span transmembrane peptides, the FXYD proteins. This diverse family of regulators has in common a domain containing a Phe-X-Tyr-Asp (FXYD) motif, two conserved glycines, and one serine residue. In humans, there are seven tissue-specific FXYD proteins that differentially modulate NKA kinetics as appropriate for each system, providing dynamic responsiveness to changing physiological conditions. Our understanding of how FXYD proteins contribute to homeostasis has benefitted from recent advances described in this review: biochemical and biophysical studies have provided insight into regulatory mechanisms, genetic models have uncovered remarkable complexity of FXYD function in integrated physiological systems, new posttranslational modifications have been identified, high-resolution structural studies have revealed new details of the regulatory interaction with NKA, and new clinical correlations have been uncovered. In this review, we address the structural determinants of diverse FXYD functions and the special roles of FXYDs in various physiological systems. We also discuss the possible roles of FXYDs in protein trafficking and regulation of non-NKA targets.

Publications by Research Area

Regulatory Interactions of Transport ATPases

Protein-protein interactions of membrane proteins are difficult to quantify by traditional biochemical approaches such as coimmunoprecipitation or SPR.  These conventional methods require detergent solubilization, which can disrupt physiologically important interactions.  To circumvent this barrier we have developed methods for quantification of the affinity of transport ATPase regulatory interactions in the native environment of the membrane in living cells.  These FRET-based assays also reveal information about the quaternary structure of the protein complex.  We have focused on P-type transporters including SERCA1a, SERCA2a, and the sodium transporter NKA.  We have discovered novel determinants of functional modulation of these transporters by peptide regulators, and quantified how these regulatory interactions are tuned by phosphorylation by PKA/PKC.

Conformational Dynamics of Transport ATPases

A major focus of the Robia lab has been the structural basis of membrane transport.  Part of this effort has involved the development and utilization of “2-color transporter” constructs, novel sensors that report changes in ATPase conformation with altered intramolecular FRET.  They have applications for structural studies and are useful for high-throughput screening of drug libraries.  By combining these new tools with advanced spectroscopic methods and molecular dynamics simulations we have been able to reveal undiscovered enzymatic substates.  We have quantified microsecond/millisecond motions and identified discrete structural elements that are important for transporter function.  These important functional determinants are not accessible to traditional structural biology approaches such as X-ray crystallography.  Together with our collaborators, we have performed high throughput screening and discovered new small molecules that alter transporter structure and function.  This ongoing project seeks drug candidates that may be useful in the development of new therapies for human disease.  We have also implemented molecular dynamics simulations in our studies of ATPase conformational changes.  Comparison of the simulation results with data from physical experiments (fluorescence spectroscopy) allows us to interpret measurements made in cellular physiological environments in the context of high resolution structural information.

Structural Mechanisms of Pathogenesis

The Robia lab has used fluorescence imaging and computational approaches for investigation of pathogenic mutations, revealing new information about the fundamental mechanisms of human diseases.  We and our collaborators have studied proteins relevant to diverse diseases including muscular dystrophy, vascular disease, heart disease, infectious disease, and cancer.

Oligomerization of Transmembrane Peptides

The activity of transport ATPases is often indirectly modulated through sequestration of regulatory partners into homooligomeric complexes.  We have used fluorescence microscopy and spectroscopy to quantify the affinity, stoichiometry, and quaternary conformation of these membrane protein oligomers.  We have developed a new method to quantify the rates of exchange of protomers from the oligomer. 

Biophysics of Protein Kinase Signaling

A key determinant of protein kinase specificity is the subcellular localization of the kinase near the target substrate.  Localization of kinases is a regulated process, and kinase activation is often accompanied by a translocation of the protein from one subcellular region to another.  We have used fluorescence microscopy observe this translocation and quantify important parameters such as the kinetics of binding/unbinding.  We have discovered novel structural elements that determine kinase targeting and rates of translocation.