Hetero-oligomeric cell wall channels (porins) of Nocardia farcinica

• Published in: Biochimica et biophysica acta 2011-06, Vol.1808 (6), p.1601-1610
• Main Authors: Kläckta, Christian, Knörzer, Philipp, Rieß, Franziska, Benz, Roland
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Blotting, Western; Cell Wall - metabolism; Cell wall channel; Cloning, Molecular; Hydrogen-Ion Concentration; Lipid bilayer membrane; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Models, Molecular; Molecular Sequence Data; MspA; Mycolic acid; N. farcinica; Nocardia - genetics; Nocardia - metabolism; Porin; Porins - chemistry; Porins - genetics; Porins - metabolism; Protein Multimerization; Protein Structure, Quaternary; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Analysis, Protein; Sequence Homology, Amino Acid
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/j.bbamem.2010.11.011

The cell wall of Nocardia farcinica contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of N. farcinica for negatively charged antibiotics. Based on partial sequencing of the protein responsible for channel formation derived from N. farcinica ATTC 3318 we were able to identify the corresponding genes (nfa15890 and nfa15900) within the known genome of N. farcinica IFM 10152. The corresponding genes of N. farcinica ATTC 3318 were separately expressed in the Escherichia coli BL21DE3Omp8 strain and the N-terminal His10-tagged proteins were purified to homogeneity using immobilized metal affinity chromatography. The pure proteins were designated NfpANHis and NfpBNHis, for N. farcinica porin A and N. farcinica porin B. The two proteins were checked separately for channel formation in lipid bilayers. Our results clearly indicate that the proteins NfpANHis and NfpBNHis expressed in E. coli could only together form a channel in lipid bilayer membranes. This means that the cell wall channel of N. farcinica is formed by a heterooligomer. NfpA and NfpB form together a channel that may structurally be related to MspA of Mycobacterium smegmatis based on amino acid comparison and renaturation procedure. [Display omitted] Prospective structure of the NfpA/NfpB oligomeric cell wall channel of Nocardia farcinica. It is assumed that four NfpA–NfpB subunits (shown in the four colors) form the cell wall channel. The channel is seen from the surface side. Side view of the prospective structure of the NfpA/NfpB oligomeric cell wall channel of Nocardia farcinica. The four colors correspond to the four NfpA–NfpB subunits of the cell wall channel. The upper side of the channel structure faces the surface of the Nocardia cell. The graphic was designed using rasmol. ► The cell wall channel of the mycolata Nocardia farcinica ATTC 3318 was subjected to Edman-degradation. ► Based on BLAST analysis six genes were identified in genome of N. farcinica IFM 10152 coding for cell wall channel. ► The six genes are organized in tandems of two genes separated only by few base pairs from one another. ► The cell wall channel of N. farcinica ATTC 3318 is formed by a heteropolymer of two subunits NfpA and NfpB. ► NfpA and NfpB form together a channel structurally related to MspA of Mycobacterium smegmatis.