scholarly journals Characterization of the Chimeric PriB-SSBc Protein

2021 ◽  
Vol 22 (19) ◽  
pp. 10854
Author(s):  
En-Shyh Lin ◽  
Yen-Hua Huang ◽  
Cheng-Yang Huang
Keyword(s):  
Replication Fork ◽  
Structural Similarity ◽  
Binding Properties ◽  
Interaction Domain ◽  
Ssdna Binding ◽  
Initiator Protein ◽  
Protein Protein Interaction ◽  
Binding Behavior ◽  
Oligomerization Domain

PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein–protein interaction domain (SSBc). Apparently, the largest difference in structure between PriB and SSB is the lack of SSBc in PriB. In this study, we produced the chimeric PriB-SSBc protein in which Klebsiella pneumoniae PriB (KpPriB) was fused with SSBc of K. pneumoniae SSB (KpSSB) to characterize the possible SSBc effects on PriB function. The crystal structure of KpSSB was solved at a resolution of 2.3 Å (PDB entry 7F2N) and revealed a novel 114-GGRQ-117 motif in SSBc that pre-occupies and interacts with the ssDNA-binding sites (Asn14, Lys74, and Gln77) in SSBn. As compared with the ssDNA-binding properties of KpPriB, KpSSB, and PriB-SSBc, we observed that SSBc could significantly enhance the ssDNA-binding affinity of PriB, change the binding behavior, and further stimulate the PriA activity (an initiator protein in the pre-primosomal step of DNA replication), but not the oligomerization state, of PriB. Based on these experimental results, we discuss reasons why the properties of PriB can be retrofitted when fusing with SSBc.

scholarly journals C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site

2014 ◽  
Vol 2014 ◽  
pp. 1-16 ◽  
Author(s):  
Yen-Hua Huang ◽  
Cheng-Yang Huang
Keyword(s):  
Binding Site ◽  
Domain Swapping ◽  
Mobility Shift ◽  
Interaction Domain ◽  
Ssdna Binding ◽  
Protein Protein Interaction ◽  
Terminal Domain ◽  
Serovar Typhimurium ◽  
Mobility Shift Assay ◽  
Oligomerization Domain

Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, including DNA replication, repair, and recombination, and is therefore essential for cell survival. Bacterial SSB consists of an N-terminal ssDNA-binding/oligomerization domain and a flexible C-terminal protein-protein interaction domain. We characterized the ssDNA-binding properties ofKlebsiella pneumoniaeSSB (KpSSB),Salmonella entericaSerovar Typhimurium LT2 SSB (StSSB),Pseudomonas aeruginosaPAO1 SSB (PaSSB), and two chimeric KpSSB proteins, namely, KpSSBnStSSBc and KpSSBnPaSSBc. The C-terminal domain of StSSB or PaSSB was exchanged with that of KpSSB through protein chimeragenesis. By using the electrophoretic mobility shift assay, we characterized the stoichiometry of KpSSB, StSSB, PaSSB, KpSSBnStSSBc, and KpSSBnPaSSBc, complexed with a series of ssDNA homopolymers. The binding site sizes were determined to be26±2,21±2,29±2,21±2, and29±2nucleotides (nt), respectively. Comparison of the binding site sizes of KpSSB, KpSSBnStSSBc, and KpSSBnPaSSBc showed that the C-terminal domain swapping of SSB changes the size of the binding site. Our observations suggest that not only the conserved N-terminal domain but also the C-terminal domain of SSB is an important determinant for ssDNA binding.

Characterization of an oligomerization domain and RNA-binding properties on rotavirus nonstructural protein NS34

Virology ◽  
1992 ◽  
Vol 190 (1) ◽  
pp. 68-83 ◽  
Author(s):  
Nora M. Mattion ◽  
Jean Cohen ◽  
Carlos Aponte ◽  
Mary K. Estes
Keyword(s):  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Properties ◽  
Oligomerization Domain

Characterization of ATP and calmodulin-binding properties of a truncated form of Bacillus anthracis adenylate cyclase

Biochemistry ◽  
1990 ◽  
Vol 29 (20) ◽  
pp. 4922-4928 ◽  
Author(s):  
Elisabeth Labruyere ◽  
Michele Mock ◽  
Daniel Ladant ◽  
Susan Michelson ◽  
Anne Marie Gilles ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Bacillus Anthracis ◽  
Truncated Form ◽  
Binding Properties ◽  
Calmodulin Binding

scholarly journals Regulation of clathrin coat assembly by Eps15 homology domain–mediated interactions during endocytosis

2012 ◽  
Vol 23 (4) ◽  
pp. 687-700 ◽  
Author(s):  
Ryohei Suzuki ◽  
Junko Y. Toshima ◽  
Jiro Toshima
Keyword(s):  
Functional Diversity ◽  
Protein Interaction ◽  
Interaction Domain ◽  
Protein Protein Interaction ◽  
Molecular Events ◽  
Biological Studies ◽  
Eh Domain ◽  
Actin Patch ◽  
Lines Of Evidence

Clathrin-mediated endocytosis involves a coordinated series of molecular events regulated by interactions among a variety of proteins and lipids through specific domains. One such domain is the Eps15 homology (EH) domain, a highly conserved protein–protein interaction domain present in a number of proteins distributed from yeast to mammals. Several lines of evidence suggest that the yeast EH domain–containing proteins Pan1p, End3p, and Ede1p play important roles during endocytosis. Although genetic and cell-biological studies of these proteins suggested a role for the EH domains in clathrin-mediated endocytosis, it was unclear how they regulate clathrin coat assembly. To explore the role of the EH domain in yeast endocytosis, we mutated those of Pan1p, End3p, or Ede1p, respectively, and examined the effects of single, double, or triple mutation on clathrin coat assembly. We found that mutations of the EH domain caused a defect of cargo internalization and a delay of clathrin coat assembly but had no effect on assembly of the actin patch. We also demonstrated functional redundancy among the EH domains of Pan1p, End3p, and Ede1p for endocytosis. Of interest, the dynamics of several endocytic proteins were differentially affected by various EH domain mutations, suggesting functional diversity of each EH domain.

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