X-ray structure analysis of a designed oligomeric miniprotein reveals a discrete quaternary architecture

Abstract

The x-ray crystal structure of an oligomeric miniprotein has been determined to a 1.2-Å resolution by means of multiwavelength anomalous diffraction phasing with selenomethionine analogs that retain the biophysical characteristics of the native peptide. Peptide 1, comprising ␣ and ␤ secondary structure elements with only 21 aa per monomer, associates as a discrete tetramer. The peptide adopts a previously uncharacterized quaternary structure in which ␣ and ␤ components interact to form a tightly packed and well defined hydrophobic core. The structure provides insight into the origins of the unusual thermal stability of the oligomer. The miniprotein shares many characteristics of larger proteins, including cooperative folding, lack of 1-anilino-8-naphthalene sulfonate binding, and limited deuterium exchange, and possesses a buried surface area typical of native proteins. O ligomerization is a fundamental strategy for generating protein structural and functional complexity in nature. Many large proteins are believed to have arisen from oligomeric precursors that, by means of gene duplication and fusion, have become one encoded protein (1). Some examples are the ribosome anti-association factor eIF6 (2), the periplasmic binding proteins (3, 4), and the eightfold ␤͞␣ barrel (5). Functionally, many proteins (6) are inactive in the monomeric state but are active as a dimer or higher-order oligomer; examples include GCN4 (7) and MCP-1 (8).We are interested in studying the structural and functional advantages conferred by protein oligomerization. We thus set out to create miniproteins that would adopt a defined oligomeric state. Miniprotein models have been used to investigate biological processes and to model larger proteins through the incorporation of catalytic or other functionality (9-11). Oligomeric miniproteins serve as minimal model systems for quaternary structure formation. Moreover, they constitute platforms for probing whether more complex oligomeric structures might ultimately support function.The strategy for oligomerization was inspired by ''domain swapping,'' or the exchange of association partners, an important evolutionary mechanism for protein oligomerization (12)(13)(14)(15). This strategy has previously been used for the design of a coiled-coil domain-swapped dimer (16). The prototypic ␤␤␣ (BBA) motif BBA5 (Fig. 1) was chosen as the building block because it represents a discretely folded and structured miniprotein motif (17,18). This motif includes interactions between secondary structural elements, thus presenting an opportunity to favor similar interactions between monomers within an oligomer. Because it has been reported that shortening the linker between domains favors domain-swapped oligomers (19, 20), we introduced a bias toward oligomerization in the BBA motif by varying the length of the 3-aa hinge region between secondary structural elements (21). Our approach for the discovery of homooligomers of peptides with ␤␤␣ supersecondary structure was based on a complementary design and screening ...

Contribution of Electrostatic Interactions, Compactness and Quaternary Structure to Protein Thermostability: Lessons from Structural Genomics of Thermotoga maritima

“…Thus, protein compactness appears quite relevant to the engineering of more thermostable proteins, as shown also by the artificial peptide BBAT1. 31,45 Different strategies to achieve thermostability could be expected among different proteins, since they have different constraints related to structure and function. Indeed, this observation has already been made many times.…”

Evolution of Quaternary Structure in a Homotetrameric Enzyme

“…11 Oligomerisation represents a fundamental strategy for generating protein structural and functional complexity. 12 Possible advantages for higher order structures include genetic economy, functional gain, structural stability, 10 and increased potential for regulation. 5 Arguments have been put forward to rationalise the selection pressures to evolve and maintain specific dimerisation interfaces in biology, 5 but no general explanation has been put forward to explain the variety of quaternary structures observed, even amongst closely related proteins.…”

Creating Functional Artificial Proteins

“…The burial of hydrophobic groups and the interaction between aromatic side-chains seemed to stabilize the three-stranded conformation [24]. Recently, the design and characterization of a homotetrameric miniprotein (BBA) was reported in which each monomer (21 residues) adopted a mixed secondary structure [25]. The compact BBA miniprotein was then redesigned to convert it into a heterotetramer [26].…”