The two tryptophans of β2-microglobulin have distinct roles in function and folding and might represent two independent responses to evolutionary pressure

Abstract

BackgroundWe have recently discovered that the two tryptophans of human β2-microglobulin have distinctive roles within the structure and function of the protein. Deeply buried in the core, Trp95 is essential for folding stability, whereas Trp60, which is solvent-exposed, plays a crucial role in promoting the binding of β2-microglobulin to the heavy chain of the class I major histocompatibility complex (MHCI). We have previously shown that the thermodynamic disadvantage of having Trp60 exposed on the surface is counter-balanced by the perfect fit between it and a cavity within the MHCI heavy chain that contributes significantly to the functional stabilization of the MHCI. Therefore, based on the peculiar differences of the two tryptophans, we have analysed the evolution of β2-microglobulin with respect to these residues.ResultsHaving defined the β2-microglobulin protein family, we performed multiple sequence alignments and analysed the residue conservation in homologous proteins to generate a phylogenetic tree. Our results indicate that Trp60 is highly conserved, whereas some species have a Leu in position 95; the replacement of Trp95 with Leu destabilizes β2-microglobulin by 1 kcal/mol and accelerates the kinetics of unfolding. Both thermodynamic and kinetic data fit with the crystallographic structure of the Trp95Leu variant, which shows how the hydrophobic cavity of the wild-type protein is completely occupied by Trp95, but is only half filled by Leu95.ConclusionsWe have established that the functional Trp60 has been present within the sequence of β2-microglobulin since the evolutionary appearance of proteins responsible for acquired immunity, whereas the structural Trp95 was selected and stabilized, most likely, for its capacity to fully occupy an internal cavity of the protein thereby creating a better stabilization of its folded state.

Energy landscapes of functional proteins are inherently risky

“…Given that an intact adaptive immune response is vital for life in all higher eukaryotes, there is immense evolutionary pressure on the sequences of both the MHC-1 heavy chain and β 2 m to ensure that they fold and bind efficiently. This explains why the sequence of β 2 m is so highly conserved from cartilaginous fish to humans 83 and why the smallest changes to its sequence that cause the isomerization of a single peptide bond can lead to aggregation and disease. In addition, the interactions between β 2 m and the MHC-1 heavy chain have coevolved in such a way as to minimize aggregation risks, although the susceptibility is ever present when the fragile fold of β 2 m is left on its own.…”

The role of conformational flexibility in β2‐microglobulin amyloid fibril formation at neutral pH

“…Although frequent transitions to the unfolded state of β 2 m are likely to allow increased kinetic sampling of aggregation‐competent conformations, we have shown here that this is not sufficient to enable nucleation of β 2 m fibril formation at neutral pH. It has been suggested that conformational flexibility is dampened by evolution to reduce the potential risk of aggregation as described above . Here we show that monomeric β 2 m displays unusually high kinetic instability for an immunoglobulin domain, presumably because the evolutionary pressure on its sequence has been to enable tight binding to the MHC heavy chain.…”

“…It has been suggested that conformational flexibility is dampened by evolution to reduce the potential risk of aggregation as described above. [52] Here we show that monomeric b 2 m displays unusually high kinetic instability for an immunoglobulin domain, presumably because the evolutionary pressure on its sequence has been to enable tight binding to the MHC heavy chain.…”

Conformational stability and dynamics in crystals recapitulate protein behaviour in solution

“…In addition, this new analysis reveals the population of solvent-exposed conformations of D76N β2m W95 side-chain, in both protein states. The position of W95 is central in β2m hydrophobic core and its mutation greatly destabilises the protein fold (21,34). It is likely that β2m conformations with an exposed W95 may be highly destabilised and may represent an important step in the foldingunfolding pathway.…”

“…The high-energy state M 1 * was suggested to be associated with protein misfolding and consequently with aggregation (16,32,33). Conversely, given the pivotal role of W95 in the stability of β2m buried hydrophobic core (21,34), one may speculate that excited state M 2 *, having a highly solvent exposed W95 residue, may be associated with protein folding-unfolding and consequently with thermodynamic stability.…”