Restructuring of the dinucleotide-binding fold in an NADP(H) sensor protein

Abstract

NAD(P) has long been known as an essential energy-carrying molecule in cells. Recent data, however, indicate that NAD(P) also plays critical signaling roles in regulating cellular functions. The crystal structure of a human protein, HSCARG, with functions previously unknown, has been determined to 2.4-A resolution. The structure reveals that HSCARG can form an asymmetrical dimer with one subunit occupied by one NADP molecule and the other empty. Restructuring of its NAD(P)-binding Rossmann fold upon NADP binding changes an extended loop to an alpha-helix to restore the integrity of the Rossmann fold. The previously unobserved restructuring suggests that HSCARG may assume a resting state when the level of NADP(H) is normal within the cell. When the NADP(H) level passes a threshold, an extensive restructuring of HSCARG would result in the activation of its regulatory functions. Immunofluorescent imaging shows that HSCARG redistributes from being associated with intermediate filaments in the resting state to being dispersed in the nucleus and the cytoplasm. The structural change of HSCARG upon NADP(H) binding could be a new regulatory mechanism that responds only to a significant change of NADP(H) levels. One of the functions regulated by HSCARG may be argininosuccinate synthetase that is involved in NO synthesis.

Fig. 1.

Crystal structure of HSCARG. (a) The tracing of HSCARG in complex with the NADP⁺ molecule. The protein is shown as ribbons, and the NADP⁺ molecule is shown as the stick model (35). The color scheme of the protein is from the N terminus (dark blue) to the C terminus (magenta). Secondary structure elements are labeled according to their positions in the protein sequence. (b) The asymmetric dimer of HSCARG with molecules I (red) binding one NADP⁺ molecule (stick model) and molecule II empty (blue).

Fig. 2.

Structural changes of HSCARG upon NADP⁺ binding. Restructuring of HSCARG NADP⁺ upon binding. The blue regions represent the conformation of HSCARG before NADP⁺ binding and the red after NADP⁺ binding. The full structure of HSCARG and the bound NADP⁺ molecule is shown as white ribbons and the stick model, respectively. The residues that define the restructured regions are labeled by their residue numbers.

Fig. 3.

Subcellular localization of HSCARG in HeLa cells. (a) Overexpressed HSCARG was detected by using monoclonal anti-flag antibody (Upper). Endogenously expressed HSCARG was detected by using polyclonal anti-HSCARG generated from rat (Lower). (b) Colocalization of HSCARG with cytokeratin in HeLa cells. In Upper, HeLa cells were transfected with an expression plasmid for Flag-tagged HSCARG and double immunofluorescent stained with polyclonal anticytokeratin and anti-Flag. In Lower, untransfected HeLa cells were double-stained with polyclonal anticytokeratin and polyclonal anti-HSCARG. (Right) The overlay image of cytokeratin and HSCARG.

Fig. 4.

Effects of NADPH/NADP⁺ levels. (a) Changes in NADPH/NADP⁺ concentrations induced redistribution of HSCARG within cells. HeLa cells were treated with 25 μM DHEA for 1 and 24 h, respectively. Immunofluorescence analysis was performed as described in Materials and Methods. Images shown represent three independent experiments. (b and c) In vivo interaction of HSCARG with ASS. 293T cells were cotransfected with expression plasmids for Flag-tagged HSCARG and HA-tagged ASS. Cell extracts were prepared after 48 h and immunoprecipitated with an anti-Flag monoclonal antibody (lane 3) in b or an anti-HA monoclonal antibody (lane 6) in c or control IgG (lanes 2 and 5). Western blot analysis was performed with anti-HA antibody (b) or anti-Flag antibody (c) followed by an anti-mouse IgG secondary step that also recognizes the IgG heavy chain. Expression of HSCARG and ASS was confirmed by Western blot analyses of the cell lysates with anti-Flag antibody (c, lane 4) and anti-HA antibody (b, lane 1), respectively. IP, immunoprecipitation.