Sarco(endo)plasmic reticulum Ca 2؉ ATPase (SERCA) Ca 2؉ transporters pump cytosolic Ca 2؉ into the endoplasmic reticulum, maintaining a Ca 2؉ gradient that controls vital cell functions ranging from proliferation to death. To meet the physiological demand of the cell, SERCA activity is regulated by adjusting the affinity for Ca 2؉ ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest Ca 2؉ affinity because of a unique C-terminal extension (2b-tail). Here, an extensive structure-function analysis of SERCA2b mutants and SERCA1a2b chimera revealed how the 2b-tail controls Ca 2؉ affinity. Its transmembrane (TM) segment (TM11) and luminal extension functionally cooperate and interact with TM7/TM10 and luminal loops of SERCA2b, respectively. This stabilizes the Ca 2؉ -bound E1 conformation and alters Ca 2؉ -transport kinetics, which provides the rationale for the higher apparent Ca 2؉ affinity. Based on our NMR structure of TM11 and guided by mutagenesis results, a structural model was developed for SERCA2b that supports the proposed 2b-tail mechanism and is reminiscent of the interaction between the ␣-and ␤-subunits of Na ؉ ,K ؉ -ATPase. The 2b-tail interaction site may represent a novel target to increase the Ca 2؉ affinity of malfunctioning SERCA2a in the failing heart to improve contractility.endoplasmic reticulum ͉ Ca 2ϩ -ATPase ͉ ion transporter ͉ phospholamban ͉ P-type ATPase C alcium ions in the endoplasmic reticulum (ER) control cellular growth, proliferation, differentiation, and death and are maintained at high concentrations by the ubiquitous ER Ca 2ϩ pump sarco(endo)plasmic reticulum Ca 2ϩ ATPase 2b (SERCA2b) (1). SERCAs cycle between an E1 conformation, with high-affinity Ca 2ϩ -binding sites facing the cytoplasm, and E2 with low-affinity Ca 2ϩ -binding sites facing the lumen of the ER (2, 3). Several 3D crystal structures of SERCA1a, the related fast-twitch skeletal-muscle isoform, provided detailed insights into the mechanism of Ca 2ϩ transport. Conformational changes in the cytosolic domain of the pump are driven by ATP hydrolysis and control the opening and closing of the Ca 2ϩ gates in the transmembrane (TM) domain. The TM region contains 10 TM helices comprising residues that reversibly coordinate Ca 2ϩ for transport (4-7) (reviewed in ref. 3).Unfortunately, much less is known about the structure and mechanism of the ubiquitous SERCA2 isoform (Ϸ84% sequence identity with SERCA1a). Alternative splicing of the SERCA2 messenger yields two variants: SERCA2a, which populates the sarcoplasmic reticulum (SR) of the heart, smooth, and slow-twitch skeletal muscle; and the housekeeping variant SERCA2b, which is present in the ER of all cell types (Fig. S1a) (8). SERCA2b differs from the muscle isoforms SERCA1a or SERCA2a, displaying a 2-fold higher affinity for Ca 2ϩ and lower catalytic turnover rate. These unique functional properties are related to an extended C terminus of 49 residues (2b-tail) containing an 11th TM domain (TM11) and luminal extension (LE) (Fig. S1b) (9-12), b...

DNA binding as well as ligand binding by nuclear receptors has been studied extensively. Both binding functions are attributed to isolated domains of which the structure is known. The crystal structure of a complete receptor in complex with its ligand and DNA-response element, however, has been solved only for the peroxisome proliferator-activated receptor ␥ (PPAR␥)-retinoid X receptor ␣ (RXR␣) heterodimer. This structure provided the first indication of direct interactions between the DNA-binding domain (DBD) and ligand-binding domain (LBD). In this study, we investigated whether there is a similar interface between the DNA-and ligand-binding domains for the androgen receptor (AR). Despite the structural differences between the AR-and PPAR␥-LBD, a combination of in silico modeling and docking pointed out a putative interface between AR-DBD and AR-LBD. The surfaces were subjected to a point mutation analysis, which was inspired by known AR mutations described in androgen insensitivity syndromes and prostate cancer. Surprisingly, AR-LBD mutations D695N, R710A, F754S, and P766A induced a decrease in DNA binding but left ligand binding unaffected, while the DBD-residing mutations K590A, K592A, and E621A lowered the ligand-binding but not the DNA-binding affinity. We therefore propose that these residues are involved in allosteric communications between the AR-DBD and AR-LBD. N uclear receptors (NRs) are involved in many physiological processes, diseases, and therapeutic applications. They are transcription factors that contain a DNA-binding domain (DBD) composed of 2 zinc fingers (40) and a ligand-binding domain (LBD) formed by 12 ␣ helices (60). The structures of the separate DNA-binding and ligand-binding domains of many receptors have already revealed a large amount of information. The structure of the LBD has especially led to a more focused search for new agonists and antagonists for many therapeutic indications where NRs are involved. The exact structure of full-size NRs bound to DNA and ligand will help us in understanding the basic mechanism of nuclear receptor signaling but will also provide new targets for therapeutic strategies. The coordinates of the peroxisome proliferator-activated receptor ␥ (PPAR␥)-retinoid X receptor ␣ (RXR␣) cocrystal bound as a heterodimer to the DNA have been reported (11) and revealed a contact between PPAR␥ and RXR␣ more intimate than expected. The existence of the previously unknown interface between PPAR␥-LBD and RXR␣-DBD was corroborated with a mutation analysis. Since this has not yet been confirmed by other techniques like small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) (49), it is debated whether such a communication exists. There is, however, strong evidence for allosteric communications, e.g., between the DBD and LBD, in nuclear receptors. A most remarkable observation was made for the glucocorticoid receptor (GR) when slightly different response elements were tested in gene reporter assays: small changes in the DNA sequence had an importa...

Background:The size and/or protein nature of current von Willebrand factor (VWF)-glycoprotein (GP) Ib␣ inhibitors limits oral bioavailability and clinical development. Results: Through a rational approach, a small molecule was selected that modulates the VWF-GPIb interaction. Conclusion: Further chemical modifications will now allow full characterization and manipulation of the specific activity of the compound. Significance: Rational design allows for the identification of small molecules that interfere with protein-protein interactions.

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