Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca2+ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na+ or Ca2+ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca2+ or Na+ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.

• MeSH
• Sodium-Calcium Exchanger * chemistry   MeSH
• Mammals * MeSH
• Protein Structure, Secondary MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Sodium-Calcium Exchanger * MeSH

General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-interaction domain (CTID) in TAF13, which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.

• Keywords
• S. cerevisiae, TBP associated factors, TFIID, biochemistry, biophysics, core promoter DNA, gene regulation, histone fold domain, human, structural biology, transcription factors,
• MeSH
• DNA metabolism   MeSH
• TATA-Binding Protein Associated Factors chemistry   metabolism   MeSH
• Histone Acetyltransferases metabolism   MeSH
• Mass Spectrometry MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Protein Interaction Mapping MeSH
• Promoter Regions, Genetic MeSH
• TATA-Box Binding Protein chemistry   metabolism   MeSH
• Transcription Factor TFIID chemistry   metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• TATA-Binding Protein Associated Factors MeSH
• Histone Acetyltransferases MeSH
• TATA-Box Binding Protein MeSH
• TAF11 protein, human MeSH Browser
• TAF13 protein, human MeSH Browser
• TATA-binding protein associated factor 250 kDa MeSH Browser
• TBP protein, human MeSH Browser
• Transcription Factor TFIID MeSH

Na+/Ca2+ exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na+ or Ca2+ specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.

• Keywords
• calcium, calcium transport, calcium-binding protein, exchanger, hydrogen exchange mass spectrometry, membrane protein, membrane transport, sodium-calcium exchange, transporter,
• MeSH
• Apoproteins chemistry   genetics   metabolism   MeSH
• Archaeal Proteins chemistry   genetics   metabolism   MeSH
• Cell Membrane chemistry   MeSH
• Cysteine chemistry   MeSH
• Protein Interaction Domains and Motifs MeSH
• Mutagenesis, Insertional MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Ligands MeSH
• Methanocaldococcus metabolism   MeSH
• Models, Molecular * MeSH
• Mutation MeSH
• Peptide Fragments chemistry   genetics   metabolism   MeSH
• Sodium-Calcium Exchanger chemistry   genetics   metabolism   MeSH
• Recombinant Proteins chemistry   metabolism   MeSH
• Sodium metabolism   MeSH
• Protein Stability MeSH
• Amino Acid Substitution MeSH
• Calcium metabolism   MeSH
• Binding Sites MeSH
• Deuterium Exchange Measurement MeSH
• Computational Biology MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Apoproteins MeSH
• Archaeal Proteins MeSH
• Cysteine MeSH
• Ligands MeSH
• Peptide Fragments MeSH
• Sodium-Calcium Exchanger MeSH
• Recombinant Proteins MeSH
• Sodium MeSH
• Calcium MeSH