Double-mutant cycles: a powerful tool for analyzing protein structure and function

How T-cell receptors (TCRs) can be intrinsically biased toward MHC proteins while simultaneously display the structural adaptability required to engage diverse ligands remains a controversial puzzle. We addressed this by examining αβ TCR sequences and structures for evidence of physicochemical compatibility with MHC proteins. We found that human TCRs are enriched in the capacity to engage a polymorphic, positively charged "hot-spot" region that is almost exclusive to the α1-helix of the common human class I MHC protein, HLA-A*0201 (HLA-A2). TCR binding necessitates hot-spot burial, yielding high energetic penalties that must be offset via complementary electrostatic interactions. Enrichment of negative charges in TCR binding loops, particularly the germ-line loops encoded by the TCR Vα and Vβ genes, provides this capacity and is correlated with restricted positioning of TCRs over HLA-A2. Notably, this enrichment is absent from antibody genes. The data suggest a built-in TCR compatibility with HLA-A2 that biases receptors toward, but does not compel, particular binding modes. Our findings provide an instructional example for how structurally pliant MHC biases can be encoded within TCRs.T-cell receptor | peptide/MHC | structure | binding | MHC bias

Section: Methodsmentioning

confidence: 92%

mentioning

confidence: 99%

How structural adaptability exists alongside HLA-A2 bias in the human αβ TCR repertoire

Blevins

Pierce

Singh

et al. 2016

“…To further test whether the -clamp site has unfoldase activity, we used double-mutant cycle analysis (18), where mutations at the -clamp site could be analyzed in the context of destabilizing mutations in the substrate protein. If the clamp has a role in substrate unfolding, then destabilized LF N mutants should complement a defective -clamp mutant, and a negative interaction energy (⌬⌬G int ) should be measured.…”

Section: Role Ofmentioning

confidence: 99%

Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation

Thoren¹,

Worden

Yassif

et al. 2009

125

Cellular compartmentalization requires machinery capable of translocating polypeptides across membranes. In many cases, transported proteins must first be unfolded by means of the proton motive force and/or ATP hydrolysis. Anthrax toxin, which is composed of a channel-forming protein and two substrate proteins, is an attractive model system to study translocation-coupled unfolding, because the applied driving force can be externally controlled and translocation can be monitored directly by using electrophysiology. By controlling the driving force and introducing destabilizing point mutations in the substrate, we identified the barriers in the transport pathway, determined which barrier corresponds to protein unfolding, and mapped how the substrate protein unfolds during translocation. In contrast to previous studies, we find that the protein's structure next to the signal tag is not rate-limiting to unfolding. Instead, a more extensive part of the structure, the amino-terminal ␤-sheet subdomain, must disassemble to cross the unfolding barrier. We also find that unfolding is catalyzed by the channel's phenylalanine-clamp active site. We propose a broad molecular mechanism for translocation-coupled unfolding, which is applicable to both soluble and membrane-embedded unfolding machines.unfolding pathway ͉ transition state structure ͉ mechanical unfolding F olded proteins are Ϸ5-10 kcal⅐mol Ϫ1 more stable than their unfolded states. Therefore, the disassembly and translocation of folded proteins often require a molecular machine and a source of free energy. These ubiquitous multiprotein complexes include soluble degradation machinery, such as the proteasome or the Clp bacterial proteases (1), which unfold and degrade proteins, and some, but not all, membrane-embedded translocase channels, which can unfold and transport proteins across membranes (2). There are general features shared between these soluble and membrane-embedded translocase machines: a narrow central pore first engages the protein substrate on its free end, the substrate is unfolded mechanically, and the unfolded chain is translocated through the narrow pore, allowing it ultimately to either cross a membrane or enter into a proteolytic complex for degradation. Protein unfolding and translocation in these systems are often driven by ATP hydrolysis (1, 2), a membrane potential (⌬⌿) (2, 3), and/or a proton gradient (⌬pH) (4). The molecular mechanism of translocation-coupled unfolding, however, is poorly understood.Prior studies examining the correlation between substrate protein stability and translocation kinetics have produced conflicting results. Some ligand-stabilized substrates translocate inefficiently, because they are too thermodynamically stable (5); however, other substrates show little change in the rate of translocation when destabilized by mutagenesis (6, 7). To resolve these conflicting results, it was proposed that translocation-coupled unfolding (6, 8) depends on the mechanical stability of the local structure adjacent to the signal tag. O...

“…This equation, formally similar to the analysis of doublemutant cycles (19,26), implies that the measured coupling free energy for each mutant in the absence and presence of 0.5 M NaCl is equal to the difference in coupling energy between the native and the denatured states. If we assume that the free energy of the native state in high salt solutions is unlikely to be affected by conservative mutations mostly involving buried hydrophobic side-chains (27), ðΔΔG 0→0.…”

Section: Resultsmentioning

confidence: 98%

Deciphering the folding transition state structure and denatured state properties of Nucleophosmin C-terminal domain

Scaloni

Federici

Brunori

et al. 2010

Nucleophosmin (NPM1), one of the most abundant nucleolar proteins, is a frequent target of oncogenic mutations in acute myeloid leukaemia (AML). Mutation-induced changes at the C-terminal domain of NPM1 (Cter-NPM1) compromise its stability and cause the aberrant translocation of NPM1 to the cytosol. Hence, this protein represents a suitable candidate to investigate the relations between folding and disease. Since Cter-NPM1 folds via a compact denatured state, stabilization of the folded state of the mutated variants demands detailed structural information on both the native and denatured states. Here, we present the characterization of the complete folding pathway of Cter-NPM1 and provide molecular details for both the transition and the denatured states. The structure of the transition state was assessed by Φ-value analysis, whereas residual structure in the denatured state was mapped by evaluating the effect of mutations as modulated by conditions promoting denatured state compaction. Data reveal that folding of Cter-NPM1 proceeds via an extended nucleus and that the denatured state retains significant malleable structure at the interface between the second and third helices. Our observations constitute the essential prerequisite for structure-based drug-design studies, aimed at identifying molecules that may rescue pathological NPM1 mutants by stabilizing the native-like state.kinetics | mutagenesis | protein folding N ucleophosmin (NPM1) is a ubiquitously expressed protein that belongs to the nucleoplasmin family of nuclear chaperones and is one of the most represented nucleolar proteins (1). NPM1 is a key component of several cellular processes, ranging from ribosome biogenesis, control of centrosome duplication, maintenance of genomic stability, and cell-cycle regulation also by its direct interaction with the tumor suppressors p53 and p14arf (2). A distinctive feature of NPM1 resides in its property to rapidly shuttle from the nucleus to the cytoplasm and backwards (3), a property that may require some structural malleability.The NPM1 gene has been found overexpressed in several solid human cancers and is also a frequent target of translocations occurring in hematopoietic tumors. Mutation of the NPM1 gene is the most frequent genetic lesion in acute myeloid leukemia (AML) displaying a normal kariotype (4). Such mutations map at the C-terminal domain of the protein, and all result in its denaturation and appearance of an additional nuclear export signal. These two signatures concur and are both necessary to cause the stable and aberrant cytoplasmic localization of NPM1 mutants (1). Furthermore, NPM1 mutants enhance the translocation in the cytoplasm of several interacting partners, including the tumor suppressor p14arf and the c-Myc E3-ubiquitin ligase Fbw7-γ (5, 6). It has been proposed that this feature is involved in the mechanism for the oncogenic properties of NPM mutants (7).Recent work (8) has shown that some pathological mutations affect the stability of proteins, yielding partial or total denatura...