Structural studies of the NOD module of the transcriptional activator MalT in complex with its regulatory proteins MalK, MalY, and Aes
Structural Biology
Final Report Abstract
Uptake and metabolism of α1–4-linked glucose polymers in E. coli is accomplished by the maltose system, comprising ten mal genes that are controlled by the transcriptional activator MalT. Two operons encode the maltose/maltodextrin transport machinery: LamB, MalE, MalF, MalG, and MalK, as well as MalM, a periplasmic protein of yet unknown function. The other three operons encode the enzymes MalS, MalP, MalQ and MalZ. MalT belongs to the class of Signal Transduction ATPases with Numerous Domains (STAND) and acts as regulatory switch in response to positive signals, such as ATP and maltotriose, as well as negative signals, mediated by the interaction with the cytoplasmic proteins MalK, MalY, and Aes. Objectives of this research project were the structural characterization of the Nucleotide-binding-Oligomerization Domain (NOD), the regulatory core of MalT, alone and/or in complex with its protein inhibitors MalK, MalY or Aes and thus to elucidate the mechanistic basis of its regulation. A total of 70 different MalT constructs were screened for expression, optimal buffer conditions and purification to obtain suitable protein preparations for crystallization. With the most promising candidate fragments, either alone or in complex with Aes or MalY, more than 24500 unique crystallization setups were analyzed. Nevertheless, after almost two years of tedious screening of expression, purification and crystallization conditions for MalT fragments, we did not obtain any crystals of the MalT NOD part. At that point the focus of the project was amended to enable the PhD student to finish his thesis with structural focus. To this end, two MalT-regulated proteins MalQ and MalM were selected. MalQ converts short maltodextrins, including maltose and maltotriose, to longer maltodextrins and glucose. E. coli strains lacking MalQ cannot grow on maltose and maltotriose. We have determined three MalQ structures: apo, in complex with maltose and in complex with the pseudoheptasaccharide inhibitor acarviosine-glucose-acarbose at resolutions down to 2.1 Å. An enzymatic MalQ assay revealed the dependence between maltodextrin substrate length and product spectrum. To determine the structure of MalM, a periplasmic of unknown function, we have generated ~30 expression constructs, of which most were well soluble and could be purified to homogeneity. Best crystals of a mutated N- terminal MalM fragment diffracted only to 7 Å resolution, but the structure of a C-terminal MalM fragment could be solved at 1.8 Å resolution. Furthermore, we were able to determine the structure of apo Aes at 1.8 Å resolution and identified the Aes surface area that interacts with MalT. Protein Data Bank entries: Aes-apo (4KRX), Aes-PMS (4KRY), MalQ-apo (4S3P), MalQ•maltose (4S3Q), MalQ•acarviosine-glucose-acarbose (4S3R).
Publications
- (2014) Structural and mutational analyses of Aes, an inhibitor of MalT in Escherichia coli. Proteins, 82, 268-277
Schiefner, A., Gerber, K., Brosig, A. & Boos, W.
(See online at https://doi.org/10.1002/prot.24383) - (2015) Structural basis for the interconversion of maltodextrins by MalQ, the amylomaltase of Escherichia coli. J Biol Chem, 290, 21352-21364
Weiss, S.C., Skerra, A. & Schiefner, A.
(See online at https://doi.org/10.1074/jbc.M115.667337)
