Genome-wide analysis of KIX gene family for organ size regulation in soybean (Glycine max L.).

The KIX domain, conserved among various nuclear and co-activator factors, acts as a binding site that interacts with other transcriptional activators and co-activators, playing a crucial role in gene expression regulation. In plants, the KIX domain is involved in plant hormone signaling, stress response regulation, cell cycle control, and differentiation, indicating its potential relevance to crop productivity. This study aims to identify and characterize KIX domains within the soybean (Glycine max L.) genome to predict their potential role in improving crop productivity. The conservation and evolutionary history of the KIX domains were explored in 59 plant species, confirming the presence of the KIX domains in diverse plants. Specifically, 13 KIX domains were identified within the soybean genome and classified into four main groups, namely GmKIX8/9, GmMED15, GmHAC, and GmRECQL, through sequence alignment, structural analysis, and phylogenetic tree construction. Association analysis was performed between KIX domain haplotypes and soybean seed-related agronomic traits using re-sequencing data from a core collection of 422 accessions. The results revealed correlations between SNP variations observed in GmKIX8-3 and GmMED15-4 and soybean seed phenotypic traits. Additionally, transcriptome analysis confirmed significant expression of the KIX domains during the early stages of soybean seed development. This study provides the first characterization of the structural, expression, genomic haplotype, and molecular features of the KIX domain in soybean, offering a foundation for functional analysis of the KIX domain in soybean and other plants.

Identification of novel inhibitors against Med15a KIX domain of Candida glabrata.

Candida glabrata, the second most common cause of invasive fungal infections, exhibits multi-drug resistance to commonly used antifungal drugs. To counter this resistance, there is a critical need for novel antifungals. This study identifies small molecule inhibitors that target a three-helix bundle KIX domain in the Med15a Mediator subunit of Candida glabrata (CgMed15a KIX). This domain plays a crucial role by interacting with the Pleiotropic Drug Resistance transcription factor Pdr1, a key regulator of the multidrug resistance pathway in Candida glabrata. We performed high throughput computational screening of large chemical datasets against the binding sites of the CgMed15a KIX domain to identify novel inhibitors. We selected six potential candidates with high affinity and confirmed their binding with the CgMed15a KIX domain. A phytochemical compound, Chebulinic acid binds to the CgMed15a KIX domain with a KD value of 0.339 µM and shows significant inhibitory effects on the growth of Candida glabrata. Molecular dynamics simulation studies further revealed the structural stability of the CgMed15a KIX-Chebulinic acid complex. Thus, in conclusion, this study highlights Chebulinic acid as a novel potential antifungal compound against Candida glabrata.

Assessing the applicability of 19F labeled tryptophan residues to quantify protein dynamics.

Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to study the dynamics of biomolecules in solution. Most NMR studies exploit the spins of proton, carbon and nitrogen isotopes, as these atoms are highly abundant in proteins and nucleic acids. As an alternative and complementary approach, fluorine atoms can be introduced into biomolecules at specific sites of interest. These labels can then be used as sensitive probes for biomolecular structure, dynamics or interactions. Here, we address if the replacement of tryptophan with 5-fluorotryptophan residues has an effect on the overall dynamics of proteins and if the introduced fluorine probe is able to accurately report on global exchange processes. For the four different model proteins (KIX, Dcp1, Dcp2 and DcpS) that we examined, we established that 15N CPMG relaxation dispersion or EXSY profiles are not affected by the 5-fluorotryptophan, indicating that this replacement of a proton with a fluorine has no effect on the protein motions. However, we found that the motions that the 5-fluorotryptophan reports on can be significantly faster than the backbone motions. This implies that care needs to be taken when interpreting fluorine relaxation data in terms of global protein motions. In summary, our results underscore the great potential of fluorine NMR methods, but also highlight potential pitfalls that need to be considered.

The CBP KIX domain regulates long-term memory and circadian activity.

BACKGROUND: CREB-dependent transcription necessary for long-term memory is driven by interactions with CREB-binding protein (CBP), a multi-domain protein that binds numerous transcription factors potentially affecting expression of thousands of genes. Identifying specific domain functions for multi-domain proteins is essential to understand processes such as cognitive function and circadian clocks. We investigated the function of the CBP KIX domain in hippocampal memory and gene expression using CBPKIX/KIX mice with mutations that prevent phospho-CREB (Ser133) binding. RESULTS: We found that CBPKIX/KIX mice were impaired in long-term memory, but not learning acquisition or short-term memory for the Morris water maze. Using an unbiased analysis of gene expression in the dorsal hippocampus after training in the Morris water maze or contextual fear conditioning, we discovered dysregulation of CREB, CLOCK, and BMAL1 target genes and downregulation of circadian genes in CBPKIX/KIX mice. Given our finding that the CBP KIX domain was important for transcription of circadian genes, we profiled circadian activity and phase resetting in CBPKIX/KIX mice. CBPKIX/KIX mice exhibited delayed activity peaks after light offset and longer free-running periods in constant dark. Interestingly, CBPKIX/KIX mice displayed phase delays and advances in response to photic stimulation comparable to wildtype littermates. Thus, this work delineates site-specific regulation of the circadian clock by a multi-domain protein. CONCLUSIONS: These studies provide insight into the significance of the CBP KIX domain by defining targets of CBP transcriptional co-activation in memory and the role of the CBP KIX domain in vivo on circadian rhythms.

Structural and mechanistic insights into the interaction of the circadian transcription factor BMAL1 with the KIX domain of the CREB-binding protein.

The mammalian CLOCK:BMAL1 transcription factor complex and its coactivators CREB-binding protein (CBP)/p300 and mixed-lineage leukemia 1 (MLL1) critically regulate circadian transcription and chromatin modification. Circadian oscillations are regulated by interactions of BMAL1's C-terminal transactivation domain (TAD) with the KIX domain of CBP/p300 (activating) and with the clock protein CRY1 (repressing) as well as by the BMAL1 G-region preceding the TAD. Circadian acetylation of Lys537 within the G-region enhances repressive BMAL1-TAD-CRY1 interactions. Here, we characterized the interaction of the CBP-KIX domain with BMAL1 proteins, including the BMAL1-TAD, parts of the G-region, and Lys537 Tethering the small compound 1-10 in the MLL-binding pocket of the CBP-KIX domain weakened BMAL1 binding, and MLL1-bound KIX did not form a ternary complex with BMAL1, indicating that the MLL-binding pocket is important for KIX-BMAL1 interactions. Small-angle X-ray scattering (SAXS) models of BMAL1 and BMAL1:KIX complexes revealed that the N-terminal BMAL1 G-region including Lys537 forms elongated extensions emerging from the bulkier BMAL1-TAD:KIX core complex. Fitting high-resolution KIX domain structures into the SAXS-derived envelopes suggested that the G-region emerges near the MLL-binding pocket, further supporting a role of this pocket in BMAL1 binding. Additionally, mutations in the second CREB-pKID/c-Myb-binding pocket of the KIX domain moderately impacted BMAL1 binding. The BMAL1(K537Q) mutation mimicking Lys537 acetylation, however, did not affect the KIX-binding affinity, in contrast to its enhancing effect on CRY1 binding. Our results significantly advance the mechanistic understanding of the protein interaction networks controlling CLOCK:BMAL1- and CBP-dependent gene regulation in the mammalian circadian clock.

KIX domain of AtMed15a, a Mediator subunit of Arabidopsis, is required for its interaction with different proteins.

Med15 is an important subunit of Mediator Tail module and is characterized by a KIX domain present towards amino terminal. In yeast and metazoans, Med15 KIX domain has been found to interact with various transcription factors regulating several processes including carbohydrate metabolism, lipogenesis, stress response and multidrug resistance. Mechanism of Med15 functioning in Arabidopsis is largely unknown. In this study, interactome of KIX domain of Arabidopsis Med15, AtMed15a, was characterized. We found 45 proteins that interact with AtMed15a KIX domain, including 11 transcription factors, 3 single strand nucleic acid-binding proteins and 1 splicing factor. The third helix of the KIX domain was found to be involved in most of the interactions. Mapping of the regions participating in the interactions revealed that the activation domain of a transcription factor, UKTF1 interacted with AtMed15a KIX domain. Thus, our results suggest that in Arabidopsis, activation domain of transcription factors target KIX domain of AtMed15a for their transcriptional responses.

Kix domain specific Immunoglobulin A can protect from adverse lung and cerebral pathology induced by Plasmodium berghei ANKA.

Plasmodium specific IgA has been detected in serum and breast milk among the endemic population but the role it can play in vivo is not clear. In this report, we demonstrate the utility of Malaria specific IgA, elicited by peptide sequences (referred as Mpep3 and Mpep4) of region VI of EBA-175 (PfrVI). Immunization of mice with KLH tagged or untagged peptides of Mpep3, Mpep4 or with PfrVI have resulted in specific IgA response that inhibits the in vitro invasion of Plasmodium falciparum merozoites. Mice having the IgA specific to Mpep4 have exhibited higher tolerance to Plasmodium berghei ANKA parasitemia, exhibited several fold lesser sequestration of infected RBC, lesser damage to microvasculature with no signs of perivascular haemorrhage and lesser lung inflammation in comparison to unimmunized mice. In addition, the immunized mice have B-cell population that secrete the IgA specific to PfrVI. These results suggest that the IgA specific to these malarial antigens can confer significant advantage to hosts and it may also reduce the severity of malaria infection.