Crystal structures of MBP fusion proteins

Abstract

Although chaperone-assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose-binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter-domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP-mediated structural distortions are very rare.

Design of an expression system to enhance MBP-mediated crystallization

“…The structure of the hNLRP1-CARD was determined previously using a selenomethionine-labeled form at 3.1 Å resolution (3KAT). Because essentially identical structures were determined using our MBP-mediated crystallization strategy, and we observed minimal interactions between the MBP tag and the targets in our current studies, our data suggest that fusion protein tags such as MBP are unlikely to interfere with the structure or folding of death domains, and maybe other protein targets as reported previously137. Even though we have only tested crystallization of the death domain superfamily members, our MBP fusion strategy may promote crystallization of diverse protein targets, as was demonstrated by previously determined structures of MBP fused with targets of various sizes or folds.…”

“…In agreement with a recent review ref. 37, we have not been able to find consensus linker sequence in these structures, although a linker with the sequence AAA appears to be common.…”

“…There are over 100 deposited MBP fusion protein structures in the PDB by the middle of 2016, making MBP one of the most utilized crystallization chaperone (also see Waugh’s recent review ref. 37). The MBP-tagged targets vary in their overall folds, function, and origins (Table S1).…”

“…To explore the method of fusion tag-mediated crystallization, the biggest challenge is still the design of such chimeric expression constructs to maximize the crystallizability of the targets18. Waugh stated in a recent review on crystal structures of MBP fusion proteins that a short helical linker (i.e., NAAA) was a frequently used linker37. Despite that using MBP tag as an expression and crystallization platform is increasingly appreciated3839, the optimal sequence, length, and structure of the linker between MBP and the target proteins have not been fully investigated, which limits the application of this crystallization strategy.…”

The macro domain as fusion tag for carrier‐driven crystallization

“…Carrier‐driven crystallization, or chaperone‐assisted crystallization, describes the crystallization of a target protein by fusing it to a well‐behaving protein tag, which may contribute to the formation of the crystal lattice. A small selection of well‐characterized proteins has thus far been used as crystallization tags: Short fragments of fibrinogen and α‐actin have been crystallized by fusing them to E. coli glutathione‐S‐transferase (GST) or the catalytic domain of myosin II, respectively.…”