RESUMO
The ParABS system, composed of ParA (an ATPase), ParB (a DNA binding protein), and parS (a centromere-like DNA), regulates bacterial chromosome partition. The ParB-parS partition complex interacts with the nucleoid-bound ParA to form the nucleoid-adaptor complex (NAC). In Helicobacter pylori, ParA and ParB homologs are encoded as HpSoj and HpSpo0J (HpParA and HpParB), respectively. We determined the crystal structures of the ATP hydrolysis deficient mutant, HpParAD41A, and the HpParAD41A-DNA complex. We assayed the CTPase activity of HpParB and identified two potential DNA binding modes of HpParB regulated by CTP, one is the specific DNA binding by the DNA binding domain and the other is the non-specific DNA binding through the C-terminal domain under the regulation of CTP. We observed an interaction between HpParAD41A and the N-terminus fragment of HpParB (residue 1-10, HpParBN10) and determined the crystal structure of the ternary complex, HpParAD41A-DNA-HpParBN10 complex which mimics the NAC formation. HpParBN10 binds near the HpParAD41A dimer interface and is clamped by flexible loops, L23 and L34, through a specific cation-π interaction between Arg9 of HpParBN10 and Phe52 of HpParAD41A. We propose a molecular mechanism model of the ParABS system providing insight into chromosome partition in bacteria.
Assuntos
Proteínas de Bactérias , Cromossomos Bacterianos , Proteínas de Ligação a DNA , Helicobacter pylori , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Cromossomos Bacterianos/metabolismo , Cromossomos Bacterianos/química , Cromossomos Bacterianos/genética , Modelos Moleculares , Cristalografia por Raios X , Ligação Proteica , DNA Bacteriano/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Segregação de Cromossomos , Trifosfato de Adenosina/metabolismo , Sítios de LigaçãoRESUMO
Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA-DNA and SegB-DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon-helix-helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA-SegB-DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation.
Assuntos
Proteínas Arqueais/genética , Segregação de Cromossomos , Cromossomos de Archaea/genética , DNA Arqueal/genética , Sulfolobus solfataricus/genética , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestrutura , Cristalografia por Raios X , DNA Arqueal/química , DNA Arqueal/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Sulfolobus solfataricus/metabolismoRESUMO
Inorganic phosphate (Pi) is a fundamental and essential element for nucleotide biosynthesis, energy supply, and cellular signaling in living organisms. Human phosphate transporter (hPiT) dysfunction causes numerous diseases, but the molecular mechanism underlying transporters remains elusive. We report the structure of the sodium-dependent phosphate transporter from Thermotoga maritima (TmPiT) in complex with sodium and phosphate (TmPiT-Na/Pi) at 2.3-angstrom resolution. We reveal that one phosphate and two sodium ions (Pi-2Na) are located at the core of TmPiT and that the third sodium ion (Nafore) is located near the inner membrane boundary. We propose an elevator-like mechanism for sodium and phosphate transport by TmPiT, with the TmPiT-Na/Pi complex adopting an inward occluded conformation. We found that disease-related hPiT variants carry mutations in the corresponding sodium- and phosphate-binding residues identified in TmPiT. Our three-dimensional structure of TmPiT provides a framework for understanding PiT dysfunction and for future structure-based drug design.
RESUMO
ParABS, an important DNA partitioning process in chromosome segregation, includes ParA (an ATPase), ParB (a parS binding protein) and parS (a centromere-like DNA). The homologous proteins of ParA and ParB in Helicobacter pylori are HpSoj and HpSpo0J, respectively. We analyzed the ATPase activity of HpSoj and found that it is enhanced by both DNA and HpSpo0J. Crystal structures of HpSoj and its DNA complexes revealed a typical ATPase fold and that it is dimeric. DNA binding by HpSoj is promoted by ATP. The HpSoj-ATP-DNA complex non-specifically binds DNA through a continuous basic binding patch formed by lysine residues, with a single DNA-binding site. This complex exhibits a DNA-binding adept state with an active ATP-bound conformation, whereas the HpSoj-ADP-DNA complex may represent a transient DNA-bound state. Based on structural comparisons, HpSoj exhibits a similar DNA binding surface to the bacterial ParA superfamily, but the archaeal ParA superfamily exhibits distinct non-specific DNA-binding via two DNA-binding sites. We detected the HpSpo0J-HpSoj-DNA complex by electron microscopy and show that this nucleoid-adaptor complex (NAC) is formed through HpSoj and HpSpo0J interaction and parS DNA binding. NAC formation is promoted by HpSoj participation and specific parS DNA facilitation.