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Allosteric and energetic remodeling of a PDZ domain by protein domain extensions

ABSTRACT: Many functions of proteins are performed by independently folding structural units called domains. These domains are structurally conserved but not identical in different proteins. Here, to understand the consequences for regulation and evolvability of domain extensions, we quantify the energetic effects of two extensions in a model protein domain. Quantifying abundance and ligand binding for >190,000 protein variants allows us to measure the free energy changes for mutations throughout a PDZ domain and ~7000 energetic couplings between these mutations and two domain extensions. Both a structured extension and a more dynamic extension substantially but specifically re-shape the energy landscape. In particular, deleting an ɑ-helix alters the energetic consequences of >400 mutations in >50 sites on fold stability or binding energy, and the effects of >300 allosteric mutations, including at solvent-accessible surface sites. Extending or pruning the domain therefore reshapes its energetic and allosteric landscape, adding and removing opportunities for the allosteric control of protein function.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE299757 | GEO | 2025/07/25

REPOSITORIES: GEO

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Project description:Allosteric communication between distant sites in proteins is central to nearly all biological regulation but still poorly characterised for most proteins, limiting conceptual understanding, biological engineering and allosteric drug development. Typically only a few allosteric sites are known in model proteins, but theoretical, evolutionary and some experimental studies suggest they may be much more widely distributed. An important reason why allostery remains poorly characterised is the lack of methods to systematically quantify long-range communication in diverse proteins. Here we address this shortcoming by developing a method that uses deep mutational scanning to comprehensively map the allosteric landscapes of protein interaction domains. The key concept of the approach is the use of ‘multidimensional mutagenesis’: mutational effects are quantified for multiple molecular phenotypes—here binding and protein abundance—and in multiple genetic backgrounds. This is an efficient experimental design that allows the underlying causal biophysical effects of mutations to be accurately inferred en masse by fitting thermodynamic models using neural networks. We apply the approach to two of the most common human protein interaction domains, an SH3 domain and a PDZ domain, to produce the first global atlases of allosteric mutations for any proteins. Allosteric mutations are widely dispersed with extensive long-range tuning of binding affinity and a large mutational target space of network-altering ‘edgetic’ variants. Mutations are more likely to be allosteric closer to binding interfaces, at Glycines in secondary structure elements and at particular sites including a chain of residues connecting to an opposite surface in the PDZ domain. This general approach of quantifying mutational effects for multiple molecular phenotypes and in multiple genetic backgrounds should allow the energetic and allosteric landscapes of many proteins to be rapidly and comprehensively mapped.

Project description:Mutations in genes encoding components of the telomerase holoenzyme complex result in a spectrum of rare genetic disorders known as telomere diseases, including dyskeratosis congenita (DC). A consistent finding in DC due to pathogenic mutations in DKC1, which encodes dyskerin, is decreased steady-state levels of the non-coding RNA component of telomerase (TERC) and thus impaired telomere maintenance. Dyskerin binds hundreds of other small nucleolar RNAs (snoRNAs). However, the mechanisms by which DKC1 mutations cause variable impacts on these snoRNAs are poorly understood, which is a barrier to understanding disease mechanisms in DC beyond impaired telomere maintenance. Here, using somatic and induced pluripotent stem cells (iPSCs) from DC patients with DKC1 mutations and CRISPR-Cas9-engineered iPSCs, we show that mutations in the N-terminal extension domain (NTE) of dyskerin dysregulate the biogenesis of a subset of snoRNAs, with the most prominent effect on scaRNA13 (small Cajal body-specific RNA 13). In patient iPSCs carrying the del37L dyskerin NTE-domain mutation but not in those with C-terminal mutations, nascent scaRNA13 transcripts showed a discrete population of 3´-extended forms, as seen in the setting of DC-causing mutations in the PARN (polyA-specific ribonuclease) gene. By deep sequencing of RNA 3´ ends, we found that aberrant scaRNA13 transcripts were composed of genomically-encoded extensions and post-transcriptionally oligoadenylated species, mediated by the noncanonical polymerase PAPD5, which counters PARN. NTE domain mutations generated using CRISPR-Cas9 engineering of the endogenous DKC1 recapitulated the scaRNA13 3´-end processing defects seen in del37L patient cells. Conversely, repair of the DKC1 del37L mutation and genetic or pharmacological manipulation of PAPD5 rescued scaRNA13 end processing defects and steady-state levels. Analysis of the human telomerase cryo-EM structure showed that the dyskerin NTE interacts with 3´ end of bound RNA, suggesting that mutations in this domain impair 3´ end protection of nascent scaRNA13 in addition to canonical functions in snoRNA stabilization. Our results provide mechanistic insights into the interplay of dyskerin and the PARN/PAPD5 axis in the biogenesis and accumulation of snoRNAs beyond TERC, which has important implications for our broader understanding of ncRNA dysregulation in human diseases.

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Allosteric and energetic remodeling of a PDZ domain by protein domain extensions

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