Protein Science最新文献

Protein-protein interactions are often mediated by a modular peptide recognition domain binding to a short linear motif (SLiM) in the disordered region of another protein. To understand the features of SLiMs that are important for binding and to identify motif instances that are important for biological function, it is useful to examine the evolutionary conservation of motifs across homologous proteins. However, the intrinsically disordered regions (IDRs) in which SLiMs reside evolve rapidly. Consequently, multiple sequence alignment (MSA) of IDRs often misaligns SLiMs and underestimates their conservation. We present PairK (pairwise k-mer alignment), an MSA-free method to align and quantify the relative local conservation of subsequences within an IDR. Lacking a ground truth for conservation, we tested PairK on the task of distinguishing biologically important motif instances from background motifs, under the assumption that biologically important motifs are more conserved. The method outperforms both standard MSA-based conservation scores and a modern LLM-based conservation score predictor. PairK can quantify conservation over wider phylogenetic distances than MSAs, indicating that some SLiMs are more conserved than MSA-based metrics imply. PairK is available as an open-source python package at https://github.com/jacksonh1/pairk. It is designed to be easily adapted for use with other SLiM tools and for diverse applications.

Human neutrophil elastase (hNE), a serine protease released by neutrophils during inflammation, plays a major role in the pathophysiology of several conditions especially in inflammatory lung diseases. Its inhibition constitutes, therefore, a promising therapeutic strategy to combat these diseases. In this work, we characterized the in vitro properties of a VHH (i.e., the antigen binding domain of camelid heavy chain-only antibodies), referred to as NbE201. This VHH is able to inhibit tightly, selectively and competitively both human and murine elastases with the inhibition constants (K_i) of 4.1 ± 0.9 nM and 36.8 ± 3.9 nM, respectively. The IC₅₀ for the inhibition of the hydrolysis of elastin is in the same range to that of alpha-1 antitrypsin (i.e., the main endogenous inhibitor of hNE also used in the clinic) and 14 times better than that of Sivelestat (i.e., the 2nd clinically approved hNE inhibitor). The X-ray crystal structure of the NbE201-hNE complex reveals that the Complementarity Determining Regions CDR1 and CDR3 of the VHH bind into the substrate binding pocket of hNE and prevent the access to small or macromolecular substrates. They do not, however, bind deep enough into the pocket to be hydrolyzed. NbE201 is highly stable towards oxidation, deamidation, and chemical or thermal denaturation. NbE201 is therefore likely to tolerate manufacturing processes during drug development. These results highlight the high potential of NbE201 as a (pre)clinical tool to diagnose and treat diseases associated with excessive hNE activity, and for fundamental research to better understand the role of hNE in these conditions.

The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis.

Small calcium-binding proteins such as parvalbumins (PVs) are major seafood and fish allergens. However, the impact of structural changes on their capacity to bind IgE has not been studied in detail. Therefore, fish and reptilian PVs, as well as human α-PV, were selected for biochemical, structural, and IgE binding studies. Likely due to their high solubility, crystallization proved difficult, so additional techniques were used to promote crystallization of the proteins. Novel crystal structures were determined for human PV, cod allergen Gad m 1.0201, saltwater crocodile allergen Cro p 1.0101, and the α-PV from thornback ray. β-PVs are considered the major fish allergens, while α-PVs are rarely categorized as allergens. To explain these differences, the results of structural and IgE binding studies were combined. This approach allowed us to provide new insight into IgE binding epitopes present on PVs, focusing on cross-reactivity among the selected α- and β-PVs. In addition, we have shown that these proteins display remarkable thermal stability across a range of pH conditions, which is relevant in the case of food allergens and food processing. Moreover, it is shown that the presence of calcium cations is critical for stability of the studied PVs via their protein folding, which has an impact on the formation of IgE binding epitopes. These studies shows the stability of fish and reptile PV allergens, and it allows for further evaluation of their IgE cross-reactivity.

The pyridoxal 5'-phosphate binding protein (PLP-BP) is believed to play a crucial role in PLP homeostasis, which may explain why it is found in living organisms from all kingdoms. Escherichia coli YggS is the most studied homolog, but human PLP-BP has also attracted much attention because variants of this protein are responsible for a severe form of B₆-responsive neonatal epilepsy. Yet, how PLP-BP is involved in PLP homeostasis, and thus what its actual function is in cellular metabolism, is entirely unknown. The present study shows that YggS binds RNA and that the strength of this interaction is modulated by PLP. A key role in RNA binding is clearly played by Lys137, an invariant residue located on a protein loop away from the PLP binding site, whose importance has been highlighted previously. The interaction with RNA is evidently conserved, since it is also observed with human PLP-BP. The RNA binding site, which is apparently located at the entrance of the PLP-binding site, is also evolutionarily conserved. It is therefore reasonable to assume that PLP, by defining the conformation of the protein, determines the RNA binding affinity. RNA-seq analysis of RNA co-purified with or captured by YggS revealed SsrA and RnpB RNAs, respectively involved in trans-translation and tRNA maturation, as the major molecular components. This work opens up new horizons for the function of the PLP-BP, which could be related to its interaction with RNA and modulated by PLP, and thus play a role in an as yet unknown regulatory mechanism.

The binding of Ca²⁺ ions within the troponin core of the cardiac thin filament (CTF) regulates normal contraction and relaxation. Mutations within the troponin complexes are known to alter normal functions and result in the eventual development of cardiomyopathy. However, despite the importance of the problem, detailed microscopic knowledge of the mechanism of pathogenic effect of point mutations and their effects on the conformational free energy surface of CTF remains elusive. Mutations are known to transmit their effects hundreds of angstroms along this protein complex and between different component proteins. To explore the impact of point mutations on the conformational free energy barrier between the closed and blocked state of CTF, and to understand the transmission of mutation, we have carried out metadynamics simulations for the wild-type (WT) and two mutants (cardiac troponin T Arg92Trp (R92W) and Arg92Leu (R92L)). Specifically, we have investigated the conformational modification of the tropomyosin (Tm) and the troponin (Tn) complex during the closed-to-blocked state transition for both the WT and two hypertrophic cardiomyopathy causing mutations. Our calculations demonstrated that mutations within the cardiac troponin T (cTnT) protein alter conformational properties of the Tm and the other proteins of the Tn complex as well as the Ca²⁺ binding affinity of the cTnC protein through the indirect mediation of cardiac troponin I (cTnI). Importantly, the data revealed a significant influence of the mutations on the conformational transition free energy barriers for both the Tm and cTnC proteins. Furthermore, we found both mutations independently alter the free energy barrier of transitions of cTnT. Such alteration in the free energy upon mutation of one protein in a complex, allosterically affects the others through structural and dynamical changes, leading to a pathogenic effect on the function of the thin filament.

Human nucleotide exchange factors GRPEL1 and GRPEL2 play pivotal roles in the ADP-ATP exchange within the protein folding cycle of mitochondrial HSP70 (mtHSP70), a crucial chaperone facilitating protein import into the mitochondrial matrix. Studies in human cells and mice have indicated that while GRPEL1 serves as an essential co-chaperone for mtHSP70, GRPEL2 has a role regulated by stress. However, the precise structural and biochemical mechanisms underlying the distinct functions of the GRPEL proteins have remained elusive. In our study, we present evidence revealing that ADP-bound mtHSP70 exhibits remarkably higher affinity for GRPEL1 compared to GRPEL2, with the latter experiencing a notable decrease in affinity upon ADP binding. Additionally, Pi assay showed that GRPEL1, but not GRPEL2, enhanced the ATPase activity of mtHSP70. Utilizing Alphafold modeling, we propose that the interaction between GRPEL1 and mtHSP70 can induce the opening of the nucleotide binding cleft of the chaperone, thereby facilitating the release of ADP, whereas GRPEL2 lacks this capability. Additionally, our findings suggest that the redox-regulated Cys87 residue in GRPEL2 does not play a role in dimerization but rather reduces its affinity for mtHSP70. Our findings on the structural and functional disparities between GRPEL1 and GRPEL2 may have implications for mitochondrial protein folding and import processes under varying cellular conditions.

Episodic mitochondrial myopathy with or without optic atrophy and reversible leukoencephalopathy (MEOAL) is a rare, orphan autosomal recessive disorder caused by mutations in ferredoxin-2 (FDX2), which is a [2Fe-2S] cluster-binding protein participating in the formation of iron-sulfur clusters in mitochondria. In this biosynthetic pathway, FDX2 works as electron donor to promote the assembly of both [2Fe-2S] and [4Fe-4S] clusters. A recently identified missense mutation of MEOAL is the homozygous mutation c.431C>T (p.P144L) described in six patients from two unrelated families. This mutation alters a highly conserved proline residue located in a loop of FDX2 that is distant from the [2Fe-2S] cluster. How this Pro to Leu substitution damages iron-sulfur cluster biosynthesis is unknown. In this work, we have first compared the structural, dynamic, cluster binding and redox properties of WT and P144L [2Fe-2S] FDX2 to have clues on how the pathogenic P144L mutation can perturb the FDX2 function. Then, we have investigated the interaction of both WT and P144L [2Fe-2S] FDX2 with its physiological electron donor, ferredoxin reductase FDXR, comparing their electron transfer efficiency and protein-protein recognition patterns. Overall, the data indicate that the pathogenic P144L mutation negatively affects the FDXR-dependent electron transfer pathway from NADPH to FDX2, thereby reducing the capacity of FDX2 in assembling both [2Fe-2S] and [4Fe-4S] clusters. Our study also provided solid molecular evidences on the functional role of the C-terminal tail of FDX2 in the electron transfer between FDX2 and FDXR.

The inner membrane associated protein of 30 kDa (IM30), a member of the endosomal sorting complex required for transport (ESCRT-III) superfamily, is crucially involved in the biogenesis and maintenance of thylakoid membranes in cyanobacteria and chloroplasts. In solution, IM30 assembles into various large oligomeric barrel- or tube-like structures, whereas upon membrane binding it forms large, flat carpet structures. Dynamic localization of the protein in solution, to membranes and changes of the oligomeric states are crucial for its in vivo function. ESCRT-III proteins are known to form oligomeric structures that are dynamically assembled from monomeric/smaller oligomeric proteins, and thus these smaller building blocks must be assembled sequentially in a highly orchestrated manner, a still poorly understood process. The impact of IM30 oligomerization on function remains difficult to study due to its high intrinsic tendency to homo-oligomerize. Here, we used molecular dynamics simulations to investigate the stability of individual helices in IM30 and identified unstable regions that may provide structural flexibility. Urea-mediated disassembly of the IM30 barrel structures was spectroscopically monitored, as well as changes in the protein's tertiary and secondary structure. The experimental data were finally compared to a three-state model that describes oligomer disassembly and monomer unfolding. In this study, we identified a highly stable conserved structural core of ESCRT-III proteins and discuss the advantages of having flexible intermediate structures and their putative relevance for ESCRT-III proteins.