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Updated: Jul 5, 2026
From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins
Published on: July 4, 2016
Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans
Shunfu Piao1, Yongbin Xu, Nam Chul Ha
1College of Pharmacy and Research Institute for Drug Development, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 609-735, Republic of Korea.
Abstract:
Periplasmic membrane-fusion proteins (MFPs) are an essential component of the multidrug efflux pump in Gram-negative bacteria. They play a crucial role in bridging the outer membrane porin TolC and two distinct types of inner membrane transporters. The MFP MacA bridges the inner membrane ABC-type multidrug transporter MacB and the outer membrane porin TolC. MacA from the pathogenic bacterium Actinobacillus actinomycetemcomitans was expressed in Escherichia coli B834 (DE3) and the recombinant protein was purified using Ni-NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified MacA protein was crystallized using the vapour-diffusion method. A MAD diffraction data set was collected to a resolution of 3.0 A at 100 K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4 A, alpha = beta = 90, gamma = 120 degrees , and contains one molecule in the asymmetric unit.
Insights
Membrane-fusion protein MacA, crucial for multidrug efflux in bacteria, was purified and crystallized. This structural study provides insights into bacterial multidrug resistance mechanisms.
Area of Science:
- Structural Biology
- Microbiology
- Biochemistry
Background:
- Periplasmic membrane-fusion proteins (MFPs) are vital for multidrug efflux pumps in Gram-negative bacteria.
- MFPs connect outer membrane porins (like TolC) with inner membrane transporters (like ABC transporters).
- The MacA protein specifically links the MacB transporter to the TolC porin.
Purpose of the Study:
- To determine the structure of the MacA protein from Actinobacillus actinomycetemcomitans.
- To understand the structural basis of MacA's role in the MacB-TolC efflux complex.
Main Methods:
- Expression and purification of recombinant MacA protein from Actinobacillus actinomycetemcomitans in Escherichia coli.
- Purification using Ni-NTA affinity, Q anion-exchange, and gel-filtration chromatography.
- Crystallization via vapor-diffusion and data collection using MAD diffraction to 3.0 Å resolution.
Main Results:
- The MacA protein was successfully purified to homogeneity.
- Crystals of MacA were obtained in space group P622.
- A MAD diffraction dataset was collected, yielding structural information.
Conclusions:
- The study reports the successful purification and crystallization of MacA.
- The obtained crystal data provides a foundation for future structural determination of the MacA protein.
- This work contributes to understanding the architecture of bacterial multidrug efflux systems.
