Abstract
In pathogenic Gram-negative bacteria, many virulence factors are secreted via the two-partner secretion pathway, which consists of an exoprotein called TpsA and a cognate outer membrane translocator called TpsB. The HMW1 and HMW2 adhesins are major virulence factors in nontypeable Haemophilus influenzae and are prototype two-partner secretion pathway exoproteins. A key step in the delivery of HMW1 and HMW2 to the bacterial surface involves targeting to the HMW1B and HMW2B outer membrane translocators by an N-terminal region called the secretion domain. Here we present the crystal structure at 1.92 A of the HMW1 pro-piece (HMW1-PP), a region that contains the HMW1 secretion domain and is cleaved and released during HMW1 secretion. Structural analysis of HMW1-PP revealed a right-handed beta-helix fold containing 12 complete parallel coils and one large extra-helical domain. Comparison of HMW1-PP and the Bordetella pertussis FHA secretion domain (Fha30) reveals limited amino acid homology but shared structural features, suggesting that diverse TpsA proteins have a common structural domain required for targeting to cognate TpsB proteins. Further comparison of HMW1-PP and Fha30 structures may provide insights into the keen specificity of TpsA-TpsB interactions.
Highlights
In pathogenic Gram-negative bacteria, many virulence factors are secreted via the two-partner secretion pathway, which consists of an exoprotein called TpsA and a cognate outer membrane translocator called TpsB
In H. influenzae strain 12, the HMW1 and HMW2 proteins are synthesized as preproproteins and undergo two discrete cleavage events during the process of maturation and surface localization, the first releasing the N-terminal signal peptide corresponding to residues 1– and the second releasing the pro-piece corresponding to residues – 441
Structure Determination—In earlier work, we found that the HMW1 pro-piece (HMW1-PP, corresponding to residues 69 – 441) mediates interaction with the HMW1B outer membrane translocator and is essential for HMW1 secretion [12, 15]
Summary
Expression, and Purification of the HMW1 Pro-piece—The native HMW1 pro-piece (HMW1-PP, residues 69 – 441) was expressed as a secreted protein by generating a DH5␣ derivative that contains pHMWB::HMWC (CamR) and pHMW11–441::HAT (Clontech) (AmpR). The plasmid pHMWB::HMWC was generated by ligating a 4.8-kb NruI fragment containing hmw1B and hmw1C from pHMW1–15 [11] into NruI-digested pACYC184 In this expression system, HMW1-PP is secreted into the cell culture supernatant. After cleavage of the GST moiety from GST::HMW1-PP using PreScission protease (GE Healthcare), SeMet-labeled HMW1-PP was further purified using an anionic exchange column (Hitrap Q) and/or a gel filtration column For both columns, SeMet-labeled HMW1-PP showed the same elution profile as the native secreted protein. Secretion Assay—To assess the portion of HMW1-PP required for secretion, we generated plasmids encoding HATtagged HMW11–361 (pHMW1–361::HAT) and HMW11–269 (pHMW1–269::HAT), using pHMW11– 441::HAT as a control These plasmids were created by ligating a DNA fragment containing 340 bp of sequence upstream of the hmw1A start codon and the appropriate coding sequence into HindIII-digested pHAT10 (Clontech) and were transformed into E. coli DH5␣ harboring pACYC-HMW1B [12]. Cell sonicates and trichloroacetic acid precipitates were resolved on SDS-PAGE gels and examined by Western analysis using an antiserum against the HAT epitope
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