A pinboard by
Huan Rui

Postdoctoral scholar, University of Chicago


Computational investigation of the chemical and structural coupling in SERCA

My research focuses on a protein called the sarco/endoplasmic reticulum calcium pump. This is a calcium ion transport protein that resides in the membrane of sarcoplasmic reticulum within muscle cells. Normally, the calcium concentration is very low in the cell matrix inside the muscle cell. It is so low that even a small change in calcium concentration can be readily felt by the cell, leading to functional consequences like muscle contraction. Having elevated calcium level inside the cell can be dangerous, because it will result in hardening of the internal cell structure and in the worst-case scenario can lead to cell death. Therefore, the calcium concentration inside the muscle cell needs to be tightly controlled. The tool the muscle cell uses to exert such control is through the sarcoplasmic reticulum. This is a cell organelle that acts as a reservoir for calcium ions. It takes in the excess calcium in the cell matrix and absorbs them into its interior using the calcium pump. The calcium concentration in the sarcoplasmic reticulum interior is much higher than that in the cell matrix. As a result, the calcium pump has to work against the concentration gradient to import the calcium ions. Mechanistically, the pump is like a molecular machine that works via an “alternating-access” mechanism. The organization of the pump structure would change back and forth between two major forms, one open to the sarcoplasmic reticulum interior and the other open to the cell matrix. It is this reorganization of the pump structure that helps bring in calcium ions into the sarcoplasmic reticulum. The energy source driving this process comes from breaking down ATP molecules. This is a chemical process that also modifies the pump as it adds a phosphate group to a single amino acid in the protein. How does such a single point chemical modification translate to the mechanical work performed by the pump? This is the question I hope to address with the current research project. I use a computational method called molecular dynamics simulations to help me answer this question. So far, the results I have show that the chemical modification changes the dynamic behavior of the pump, pushes it to complete the entire transport cycle. Further experiments and computer simulations are underway to validate the current findings. Together, these findings will help us gain a complete understanding of how these pumps work and suggest new avenues for designing therapeutics.


Selectivity of externally facing ion-binding sites in the Na/K pump to alkali metals and organic cations.

Abstract: The Na/K pump is a P-type ATPase that exchanges three intracellular Na(+) ions for two extracellular K(+) ions through the plasmalemma of nearly all animal cells. The mechanisms involved in cation selection by the pump's ion-binding sites (site I and site II bind either Na(+) or K(+); site III binds only Na(+)) are poorly understood. We studied cation selectivity by outward-facing sites (high K(+) affinity) of Na/K pumps expressed in Xenopus oocytes, under voltage clamp. Guanidinium(+), methylguanidinium(+), and aminoguanidinium(+) produced two phenomena possibly reflecting actions at site III: (i) voltage-dependent inhibition (VDI) of outwardly directed pump current at saturating K(+), and (ii) induction of pump-mediated, guanidinium-derivative-carried inward current at negative potentials without Na(+) and K(+). In contrast, formamidinium(+) and acetamidinium(+) induced K(+)-like outward currents. Measurement of ouabain-sensitive ATPase activity and radiolabeled cation uptake confirmed that these cations are external K(+) congeners. Molecular dynamics simulations indicate that bound organic cations induce minor distortion of the binding sites. Among tested metals, only Li(+) induced Na(+)-like VDI, whereas all metals tested except Na(+) induced K(+)-like outward currents. Pump-mediated K(+)-like organic cation transport challenges the concept of rigid structural models in which ion specificity at site I and site II arises from a precise and unique arrangement of coordinating ligands. Furthermore, actions by guanidinium(+) derivatives suggest that Na(+) binds to site III in a hydrated form and that the inward current observed without external Na(+) and K(+) represents cation transport when normal occlusion at sites I and II is impaired. These results provide insights on external ion selectivity at the three binding sites.

Pub.: 13 Oct '10, Pinned: 29 Jun '17

Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid.

Abstract: The Na(+)/K(+) pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na(+) from the cell and import 2K(+) per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na(+)/K(+) pump that represent the kinetics of extracellular Na(+) binding/release and Na(+) occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas, we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associated with the extracellular release of the first Na(+) from the deeply occluded state. Occlusion/deocclusion transition involves large changes in enthalpy and entropy as the ion is exposed to the external milieu for release. Binding/unbinding is substantially less costly, yet larger than predicted for the energetic cost of an ion diffusing through a permeation pathway, which suggests that ion binding/unbinding must involve amino acid side-chain rearrangements at the site.

Pub.: 07 Dec '11, Pinned: 29 Jun '17

Conformational transitions and alternating-access mechanism in the sarcoplasmic reticulum calcium pump.

Abstract: Ion pumps are integral membrane proteins responsible for transporting ions against concentration gradients across biological membranes. Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), a member of the P-type ATPases family, transports two calcium ions per hydrolyzed ATP molecule via an "alternating-access" mechanism. High-resolution crystallographic structures provide invaluable insight on the structural mechanism of the ion pumping process. However, to understand the molecular details of how ATP hydrolysis is coupled to calcium transport, it is necessary to gain knowledge about the conformational transition pathways connecting the crystallographically resolved conformations. Large-scale transitions in SERCA occur at time-scales beyond the current reach of unbiased molecular dynamics (MD) simulations. Here, we overcome this challenge by employing the string method, which represents a transition pathway as a chain-of-states linking two conformational end-points. Using a highly scalable multiscale methodology, we have determined all-atom transition pathways for three main conformational transitions responsible for the alternating-access. The present pathways provide a clear chronology of the key events underlying the active transport of calcium ions by SERCA. Important conclusions are that the conformational transition that leads to occlusion with bound ATP and calcium is highly concerted and cooperative, the phosphorylation of Asp351 causes a reorganization of the cytoplasmic domains that subsequently drives the opening of the luminal gate, and re-closing of luminal gate induces a shift in the cytoplasmic domains that subsequently enables dephosphorylation of Asp351-P. Transient residue-residue contacts during the conformational transitions predicted by the computations provide an experimental route to test the general validity of the computational pathways.

Pub.: 18 Jan '17, Pinned: 29 Jun '17