Conserved Proline Residue Research Articles

Unraveling the molecular determinants of a rare human mitochondrial disorder caused by the P144L mutation of FDX2.

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.

Cathepsins L and B target HIF1α for oxygen-independent proteolytic cleavage

The oxygen-labile transcription factor called hypoxia-inducible factor (HIF) is responsible for the cellular and organismal adaptive response to reduced oxygen availability. Deregulation of HIF is associated with the pathogenesis of major human diseases including cardiovascular disease and cancer. Under normoxia, the HIFα subunit is hydroxylated on conserved proline residues within the oxygen-dependent degradation domain (ODD) that labels HIFα for proteasome-mediated degradation. Despite similar oxygen-dependent degradation machinery acting on HIF1α and HIF2α, these two paralogs have been shown to exhibit unique kinetics under hypoxia, which suggests that other regulatory processes may be at play. Here, we characterize the protease activity found in rabbit reticulocytes that specifically cleaves the ODD of HIF1α but not HIF2α. Notably, the cleavage product is observed irrespective of the oxygen-dependent prolyl-hydroxylation potential of HIF1α, suggesting independence from oxygen. HIF1α M561T substitution, which mimics an evolutionary substitution that occurred during the duplication and divergence of HIF1α and HIF2α, diminished the cleavage of HIF1α. Protease inhibitor screening suggests that cysteine proteases cathepsins L and B preferentially cleave HIF1αODD, thereby revealing an additional layer of differential HIF regulation.

Conserved proline residues prevent dimerization and aggregation in the β-lactamase BlaC.

Evolution leads to conservation of amino acid residues in protein families. Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure. Surprisingly, proline residues that are highly conserved in class A β-lactamases were found to tolerate various substitutions without large losses in enzyme activity. We investigated the roles of three conserved prolines at positions 107, 226, and 258 in the β-lactamase BlaC from Mycobacterium tuberculosis and found that mutations can lead to dimerization of the enzyme and an overall less stable protein that is prone to aggregate over time. For the variant Pro107Thr, the crystal structure shows dimer formation resembling domain swapping. It is concluded that the proline substitutions loosen the structure, enhancing multimerization. Even though the enzyme does not lose its properties without the conserved proline residues, the prolines ensure the long-term structural integrity of the enzyme.

Molecular basis for inhibition of methane clathrate growth by a deep subsurface bacterial protein.

Methane clathrates on continental margins contain the largest stores of hydrocarbons on Earth, yet the role of biomolecules in clathrate formation and stability remains almost completely unknown. Here, we report new methane clathrate-binding proteins (CbpAs) of bacterial origin discovered in metagenomes from gas clathrate-bearing ocean sediments. CbpAs show similar suppression of methane clathrate growth as the commercial gas clathrate inhibitor polyvinylpyrrolidone and inhibit clathrate growth at lower concentrations than antifreeze proteins (AFPs) previously tested. Unlike AFPs, CbpAs are selective for clathrate over ice. CbpA3 adopts a nonglobular, extended structure with an exposed hydrophobic surface, and, unexpectedly, its TxxxAxxxAxx motif common to AFPs is buried and not involved in clathrate binding. Instead, simulations and mutagenesis suggest a bipartite interaction of CbpAs with methane clathrate, with the pyrrolidine ring of a highly conserved proline residue mediating binding by filling empty clathrate cages. The discovery that CbpAs exert such potent control on methane clathrate properties implies that biomolecules from native sediment bacteria may be important for clathrate stability and habitability.

Non-specific and specific DNA binding modes of bacterial histone, HU, separately regulate distinct physiological processes through different mechanisms.

The histone-like protein HU plays a diverse role in bacterial physiology from the maintenance of chromosome structure to the regulation of gene transcription. HU binds DNA in a sequence-non-specific manner via two distinct binding modes: (i) random binding to any DNA through ionic bonds between surface-exposed lysine residues (K3, K18, and K83) and phosphate backbone (non-specific); (ii) preferential binding to contorted DNA of given structures containing a pair of kinks (structure-specific) through conserved proline residues (P63) that induce and/or stabilize the kinks. First, we show here that the P63-mediated structure-specific binding also requires the three lysine residues, which are needed for a non-specific binding. Second, we demonstrate that substituting P63 to alanine in HU had no impact on non-specific binding but caused differential transcription of diverse genes previously shown to be regulated by HU, such as those associated with the organonitrogen compound biosynthetic process, galactose metabolism, ribosome biogenesis, and cell adhesion. The structure-specific binding also helps create DNA supercoiling, which, in turn, may influence directly or indirectly the transcription of other genes. Our previous and current studies show that non-specific and structure-specific HU binding appear to have separate functions- nucleoid architecture and transcription regulation- which may be true in other DNA-binding proteins.

Activation and signaling mechanism revealed by GPR119-Gs complex structures

Agonists selectively targeting cannabinoid receptor-like G-protein-coupled receptor (GPCR) GPR119 hold promise for treating metabolic disorders while avoiding unwanted side effects. Here we present the cryo-electron microscopy (cryo-EM) structures of the human GPR119-Gs signaling complexes bound to AR231453 and MBX-2982, two representative agonists reported for GPR119. The structures reveal a one-amino acid shift of the conserved proline residue of TM5 that forms an outward bulge, opening up a hydrophobic cavity between TM4 and TM5 at the middle of the membrane for its endogenous ligands-monounsaturated lipid metabolites. In addition, we observed a salt bridge between ICL1 of GPR119 and Gβs. Disruption of the salt bridge eliminates the cAMP production of GPR119, indicating an important role of Gβs in GPR119-mediated signaling. Our structures, together with mutagenesis studies, illustrate the conserved binding mode of the chemically different agonists, and provide insights into the conformational changes in receptor activation and G protein coupling.

Expression of Mutant Glycine Receptors in Xenopus Oocytes Using Canonical and Non-Canonical Amino Acids Reveals Distinct Roles of Conserved Proline Residues.

Pentameric ligand-gated ion channels (pLGIC) play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular domain (ECD), a transmembrane domain (TMD) and an intracellular domain. The binding of the agonist to the ECD induces a structural change that is transduced to the TMD to open the channel. Molecular details of this process are emerging, but a comprehensive understanding is still lacking. Proline (Pro) is one amino acid that has attracted much interest; its unusual features generate bends in loops and kinks and bulges in helices, which can be essential for function in some pLGICs. Here, we explore the roles of four conserved Pros in the glycine receptor (GlyR), creating substitutions with canonical and noncanonical amino acids, characterizing them using two electrode voltage clamp electrophysiology in Xenopus oocytes, and interpreting changes in receptor parameters using structural data from the open and closed states of the receptor. The data reveal that for efficient function, the Pro in the α1β1 loop is needed to create a turn and to be the correct size and shape to interact with nearby residues; the peptide bond of the Pro in the Cys-loop requires the cis conformation; and the Pros in loop A and M1 allow efficient function because of their reduced hydrogen bonding capacity. These data are broadly consistent with data from other pLGICs, and therefore likely represent the important features of these Pros in all members of the family.

Evolutionarily Conserved Proline Residues Impede the Misfolding of the Mouse Prion Protein by Destabilizing an Aggregation-competent Partially Unfolded Form

The misfolding of the prion protein has been linked to several neurodegenerative diseases. Despite extensive studies, the mechanism of the misfolding process remains poorly understood. The present study structurally delineates the role of the conserved proline residues present in the structured C-terminal domain of the mouse prion protein (moPrP) in the misfolding process. It is shown that mutation of these Pro residues to Ala leads to destabilization of the native (N) state, and also to rapid misfolding. Using hydrogen–deuterium exchange (HDX) studies coupled with mass spectrometry (MS), it has been shown that the N state of moPrP is in rapid equilibrium with a partially unfolded form (PUF2*) at pH 4. It has been shown that the Pro to Ala mutations make PUF2* energetically more accessible from the N state by stabilizing it relative to the unfolded (U) state. The apparent rate constant of misfolding is found to be linearly proportional to the extent to which PUF2* is populated in equilibrium with the N state, strongly indicating that misfolding commences from PUF2*. It has also been shown that the Pro residues restrict the boundary of the structural core of the misfolded oligomers. Overall, this study highlights how the conserved proline residues control misfolding of the prion protein by modulating the stability of the partially unfolded form from which misfolding commences.

Insight into the Roles of Proline-Rich Extensin-like Receptor Protein Kinases of Bread Wheat (Triticum aestivum L.).

Proline-rich extensin-like receptor protein kinases (PERKs) are known for their roles in the developmental processes and stress responses of many plants. We have identified 30 TaPERK genes in the genome of T. aestivum, exploring their evolutionary and syntenic relationship and analyzing their gene and protein structures, various cis-regulatory elements, expression profiling, and interacting miRNAs. The TaPERK genes formed 12 homeologous groups and clustered into four phylogenetic clades. All the proteins exhibited a typical domain organization of PERK and consisted of conserved proline residue repeats and serine-proline and proline-serine repeats. Further, the tyrosine-x-tyrosine (YXY) motif was also found conserved in thirteen TaPERKs. The cis-regulatory elements and expression profiling under tissue developmental stages suggested their role in plant growth processes. Further, the differential expression of certain TaPERK genes under biotic and abiotic stress conditions suggested their involvement in defense responses as well. The interaction of TaPERK genes with different miRNAs further strengthened evidence for their diverse biological roles. In this study, a comprehensive analysis of obtained TaPERK genes was performed, enriching our knowledge of TaPERK genes and providing a foundation for further possible functional analyses in future studies.

Two adjacent phosphorylation sites in the C-terminus of the channel’s α-subunit have opposing effects on epithelial sodium channel (ENaC) activity

How phosphorylation of the epithelial sodium channel (ENaC) contributes to its regulation is incompletely understood. Previously, we demonstrated that in outside-out patches ENaC activation by serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) was abolished by mutating a serine residue in a putative SGK1 consensus motif RXRXX(S/T) in the channel’s α-subunit (S621 in rat). Interestingly, this serine residue is followed by a highly conserved proline residue rather than by a hydrophobic amino acid thought to be required for a functional SGK1 consensus motif according to invitro data. This suggests that this serine residue is a potential phosphorylation site for the dual-specificity tyrosine phosphorylated and regulated kinase 2 (DYRK2), a prototypical proline-directed kinase. Its phosphorylation may prime a highly conserved preceding serine residue (S617 in rat) to be phosphorylated by glycogen synthase kinase 3 β (GSK3β). Therefore, we investigated the effect of DYRK2 on ENaC activity in outside-out patches of Xenopus laevis oocytes heterologously expressing rat ENaC. DYRK2 included in the pipette solution significantly increased ENaC activity. In contrast, GSK3β had an inhibitory effect. Replacing S621 in αENaC with alanine (S621A) abolished the effects of both kinases. A S617A mutation reduced the inhibitory effect of GKS3β but did not prevent ENaC activation by DYRK2. Our findings suggest that phosphorylation of S621 activates ENaC and primes S617 for subsequent phosphorylation by GSK3β resulting in channel inhibition. In proof-of-concept experiments, we demonstrated that DYRK2 can also stimulate ENaC currents in microdissected mouse distal nephron, whereas GSK3β inhibits the currents.

Evolutionarily Conserved Proline Residues Impede the Misfolding of the Mouse Prion Protein by Destabilizing an Aggregation-Competent Partially Unfolded Form

Evolutionarily Conserved Proline Residues Impede the Misfolding of the Mouse Prion Protein by Destabilizing an Aggregation-Competent Partially Unfolded Form

HIF-1α Hydroxyprolines Modulate Oxygen-Dependent Protein Stability Via Single VHL Interface With Comparable Effect on Ubiquitination Rate

The basic molecular mechanism underlying mammalian oxygen-dependent regulation of hypoxia-inducible factor (HIF) via the von Hippel-Lindau E3 ubiquitin ligase is well established. The principal step in this critical cellular process is the hydroxylation of either or both of the two conserved proline residues P402 and P564 within the oxygen-dependent degradation domain (ODD) of HIF-1α subunit via prolyl hydroxylases, which is necessary for binding VHL. However, the significance of the two prolines has remained unclear considering that only one hydroxyproline is sufficient for the recruitment of VHL. Here, we show using biophysical analyses that both hydroxyprolines bind to the same interface on VHL with similar affinity; VHL binding affinity to HIF-1α ODD remains relatively unchanged regardless of whether the ODD contains one or two hydroxyprolines; ODD with two hydroxyprolines can accommodate two VHLs; and the rate of in vitro ubiquitination of ODD with one hydroxyproline via VHL E3 ligase is comparable to the rate observed with ODD containing two hydroxyprolines. However, the two hydroxyprolines show distinct contributions to the intracellular stability of HIF-1α ODD. These results demonstrate for the first time that the graduated HIF-1α stability profile observed over a range of oxygen tension is not attributed to the binding of or ubiquitination via VHL per se, but is likely due to the preceding events such as the efficacy of oxygen-dependent prolyl hydroxylase-mediated hydroxylation of HIF-1α.

HvLEAFY controls the early stages of floral organ specification and inhibits the formation of multiple ovaries in barley.

Transition to the reproductive phase, inflorescence formation and flower development are crucial elements that ensure maximum reproductive success in a plant's life cycle. To understand the regulatory mechanisms underlying correct flower development in barley (Hordeum vulgare), we characterized the multiovary 5 (mov5.o) mutant. This mutant develops abnormal flowers that exhibit mosaic floral organs typified by multiple carpels at the total or partial expense of stamens. Genetic mapping positioned mov5 on the long arm of chromosome 2H, incorporating a region that encodes HvLFY, the barley orthologue of LEAFY from Arabidopsis. Sequencing revealed that, in mov5.o plants, HvLFY contains a single amino acid substitution in a highly conserved proline residue. CRISPR-mediated knockout of HvLFY replicated the mov5.o phenotype, suggesting that HvLFYmov5 represents a loss of function allele. In heterologous assays, the HvLFYmov5 polymorphism influenced protein-protein interactions and affinity for a putative binding site in the promoter of HvMADS58, a C-class MADS-box gene. Moreover, molecular analysis indicated that HvLFY interacts with HvUFO and regulates the expression of floral homeotic genes including HvMADS2, HvMADS4 and HvMADS16. Other distinct changes in expression differ from those reported in the rice LFY mutants apo2/rfl, suggesting that LFY function in the grasses is modulated in a species-specific manner. This pathway provides a key entry point for the study of LFY function and multiple ovary formation in barley, as well as cereal species in general.

Cotranslational prolyl hydroxylation is essential for flavivirus biogenesis.

Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.

Potential transceptor AtNRT1.13 modulates shoot architecture and flowering time in a nitrate-dependent manner

Compared with root development regulated by external nutrients, less is known about how internal nutrients are monitored to control plasticity of shoot development. In this study, we characterize an Arabidopsis thaliana transceptor, NRT1.13 (NPF4.4), of the NRT1/PTR/NPF family. Different from most NRT1 transporters, NRT1.13 does not have the conserved proline residue between transmembrane domains 10 and 11; an essential residue for nitrate transport activity in CHL1/NRT1.1/NPF6.3. As expected, when expressed in oocytes, NRT1.13 showed no nitrate transport activity. However, when Ser 487 at the corresponding position was converted back to proline, NRT1.13 S487P regained nitrate uptake activity, suggesting that wild-type NRT1.13 cannot transport nitrate but can bind it. Subcellular localization and β-glucuronidase reporter analyses indicated that NRT1.13 is a plasma membrane protein expressed at the parenchyma cells next to xylem in the petioles and the stem nodes. When plants were grown with a normal concentration of nitrate, nrt1.13 showed no severe growth phenotype. However, when grown under low-nitrate conditions, nrt1.13 showed delayed flowering, increased node number, retarded branch outgrowth, and reduced lateral nitrate allocation to nodes. Our results suggest that NRT1.13 is required for low-nitrate acclimation and that internal nitrate is monitored near the xylem by NRT1.13 to regulate shoot architecture and flowering time.

Conserved proline residues in the coiled coil–OB domain linkers of Rpt proteins facilitate eukaryotic proteasome base assembly

The proteasome is a large protease complex that degrades many different cellular proteins. In eukaryotes, the 26S proteasome contains six different subunits of the ATPases associated with diverse cellular activities family, Rpt1–Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single Rpt-like ATPases associated with diverse cellular activities ATPase, the proteasome-activating nucleotidase, which forms a trimer of dimers. A key proteasome-activating nucleotidase proline residue (P91) forms cis- and trans-peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. However, the importance of the equivalent Rpt prolines for eukaryotic proteasome assembly was unknown. Here we showed that the equivalent proline is highly conserved in Rpt2, Rpt3, and Rpt5, and loosely conserved in Rpt1, in deeply divergent eukaryotes. Although in no case was a single Pro-to-Ala substitution in budding yeast strongly deleterious to growth, the rpt5–P76A mutation decreased levels of the protein and induced a mild proteasome assembly defect. Moreover, the rpt2–P103A, rpt3–P93A, and rpt5–P76A mutations all caused synthetic defects when combined with deletions of specific proteasome base assembly chaperones. The rpt2–P103A rpt5–P76A double mutant had uniquely strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant formed aggregates that were cleared, at least in part, by Hsp42 chaperone-mediated protein quality control. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization.

TET is targeted for proteasomal degradation by the PHD-pVHL pathway to reduce DNA hydroxymethylation

Hypoxia-inducible factors are heterodimeric transcription factors that play a crucial role in a cell's ability to adapt to low oxygen. The von Hippel-Lindau tumor suppressor (pVHL) acts as a master regulator of HIF activity, and its targeting of prolyl hydroxylated HIF-α for proteasomal degradation under normoxia is thought to be a major mechanism for pVHL tumor suppression and cellular response to oxygen. Whether pVHL regulates other targets through a similar mechanism is largely unknown. Here, we identify TET2/3 as novel targets of pVHL. pVHL induces proteasomal degradation of TET2/3, resulting in reduced global 5-hydroxymethylcytosine levels. Conserved proline residues within the LAP/LAP-like motifs of these two proteins are hydroxylated by the prolyl hydroxylase enzymes (PHD2/EGLN1 and PHD3/EGLN3), which is prerequisite for pVHL-mediated degradation. Using zebrafish as a model, we determined that global 5-hydroxymethylcytosine levels are enhanced in vhl-null, egln1a/b-double-null, and egln3-null embryos. Therefore, we reveal a novel function for the PHD-pVHL pathway in regulating TET protein stability and activity. These data extend our understanding of how TET proteins are regulated and provide new insight into the mechanisms of pVHL in tumor suppression.

Single‐molecule tracking reveals that the nucleoid‐associated protein HU plays a dual role in maintaining proper nucleoid volume through differential interactions with chromosomal DNA

HU (Histone-like protein from Escherichia coli strain U93) is the most conserved nucleoid-associated protein in eubacteria, but how it impacts global chromosome organization is poorly understood. Using single-molecule tracking, we demonstrate that HU exhibits nonspecific, weak, and transitory interactions with the chromosomal DNA. These interactions are largely mediated by three conserved, surface-exposed lysine residues (triK), which were previously shown to be responsible for nonspecific binding to DNA. The loss of these weak, transitory interactions in a HUα(triKA) mutant results in an over-condensed and mis-segregated nucleoid. Mutating a conserved proline residue (P63A) in the HUα subunit, deleting the HUβ subunit, or deleting nucleoid-associated naRNAs, each previously implicated in HU's high-affinity binding to kinked or cruciform DNA, leads to less dramatically altered interacting dynamics of HU compared to the HUα(triKA) mutant, but highly expanded nucleoids. Our results suggest HU plays a dual role in maintaining proper nucleoid volume through its differential interactions with chromosomal DNA. On the one hand, HU compacts the nucleoid through specific DNA structure-binding interactions. On the other hand, it decondenses the nucleoid through many nonspecific, weak, and transitory interactions with the bulk chromosome. Such dynamic interactions may contribute to the viscoelastic properties and fluidity of the bacterial nucleoid to facilitate proper chromosome functions.

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Improving the thermostability and activity of Paenibacillus pasadenensis chitinase through semi-rational design

Chitinase is a promising biocatalyst for chitin biotransformation in the field of recalcitrant biomass degradation. Excellent catalytic performance is conducive to its commercial utilization. In this work, sequence- and structure-based semi-rational design was performed to evolve the thermostability and activity of a previously identified chitinase PpChi1 from Paenibacillus pasadenensis CS0611. After combinational mutagenesis, the mutant S244C-I319C/T259P with disulfide bond introduction and proline substitution exhibited higher specific activity at higher temperature, 26.3-fold in half-life value at 50 °C, and a 7.9 °C rise in half-inactivation temperature T1/215min compared to the wild-type enzyme. The optimal reaction temperature of the mutant was shifted from 45 °C to 52.5 °C. Molecular dynamic simulation and structure analysis confirmed that these improvements of the mutant were attributed to its stabilized folding form, possibly caused by the decreased entropy of unfolding. This work gives an initial insight into the effect of conserved proline residues in thermostable chitinases and proposes a feasible approach for improving chitinase thermostability to facilitate its application in chitin hydrolysis to valuable oligosaccharides.