Role of the reaction-structure coupling in temperature compensation of the KaiABC circadian rhythm

When the mixture solution of cyanobacterial proteins, KaiA, KaiB, and KaiC, is incubated with ATP in vitro, the phosphorylation level of KaiC shows stable oscillations with the temperature-compensated circadian period. We analyzed this temperature compensation by developing a theoretical model describing the feedback relations among reactions and structural transitions in the KaiC molecule. The model showed that the reduced structural cooperativity should weaken the negative feedback coupling among reactions and structural transitions, which enlarges the oscillation amplitude and period, explaining the observed significant period extension upon single amino-acid residue substitution. We propose that an increase in thermal fluctuations similarly attenuates the reaction-structure feedback, explaining the temperature compensation in the KaiABC clock. The model suggests that the ATPase reactions in the CI domain of KaiC affect the period depending on how the reaction rates are modulated. The KaiABC clock provides a unique opportunity to analyze how the reaction-structure coupling regulates the system-level synchronized oscillations of molecules.

Single residue substitution in protamine 1 disrupts sperm genome packaging and embryonic development in mice

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by passive electrostatics between DNA and the arginine-rich core of protamines. However, phylogenetic analysis reveals several non-arginine residues that are conserved within, but not across, species. The functional significance of these residues or post-translational modifications are poorly understood. Here, we investigated the functional role of K49, a rodent-specific lysine residue in mouse protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In vivo, an alanine substitution (P1 K49A) results in ectopic histone retention, decreased sperm motility, decreased male fertility, and in zygotes, premature P1 removal from paternal chromatin. In vitro, the P1 K49A substitution decreases protamine-DNA binding and alters DNA compaction/decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential to ensure reproductive fitness.

Structure of a C1/C4-oxidizing AA9 lytic polysaccharide monooxygenase from the thermophilic fungus Malbranchea cinnamomea

The thermophilic fungus Malbranchea cinnamomea contains a host of enzymes that enable its ability as an efficient degrader of plant biomass and that could be mined for industrial applications. This thermophilic fungus has been studied and found to encode eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), which collectively possess different substrate specificities for a range of plant cell-wall-related polysaccharides and oligosaccharides. To gain greater insight into the molecular determinants defining the different specificities, structural studies were pursued and the structure of McAA9F was determined. The enzyme contains the immunoglobulin-like fold typical of previously solved AA9 LPMO structures, but contains prominent differences in the loop regions found on the surface of the substrate-binding site. Most significantly, McAA9F has a broad substrate specificity, with activity on both crystalline and soluble polysaccharides. Moreover, it contains a small loop in a region where a large loop has been proposed to govern specificity towards oligosaccharides. The presence of the small loop leads to a considerably flatter and more open surface that is likely to enable the broad specificity of the enzyme. The enzyme contains a succinimide residue substitution, arising from intramolecular cyclization of Asp10, at a position where several homologous members contain an equivalent residue but cyclization has not previously been observed. This first structure of an AA9 LPMO from M. cinnamomea aids both the understanding of this family of enzymes and the exploration of the repertoire of industrially relevant lignocellulolytic enzymes from this fungus.

A positively charged surface patch on the pestivirus NS3 protease module plays an important role in modulating NS3 helicase activity and virus production

Pestivirus nonstructural protein 3 (NS3) is a multifunctional protein with protease and helicase activities that are essential for the virus replication. In this study, we used a combination of biochemical and genetic approaches to investigate the relationship between the positively charged patch on protease module and NS3 function. The surface patch was composed of four basic residues R50, K74 and K94 in NS3 protease domain and H24 in the structurally integrated cofactor NS4A PCS . Single residue or simultaneous four-residue substitution in the patch to alanine or aspartic acid hardly affect ATPase activity. However, the single R50, K94 or H24 residue or simultaneous four-residue substitution resulted in the apparent changes of the helicase activity and RNA-binding ability of NS3. When these mutations were introduced into a classical swine fever virus (CSFV) cDNA clone, the single K94 residue or simultaneous four-residue substitution (Qua_A or Qua_D) impaired the infectious virus production. Furthermore, the replication efficiency of the CSFV variants was partially correlated to the helicase activity of NS3 in vitro . Our results suggest that the conserved positively charged patch on the NS3 plays an important role in modulating the NS3 helicase activity in vitro and CSFV production.

Current strategic limitations of phylogenetic tools badly impact the inference of an evolutionary tree

For drawing an evolutionary relationship among several protein sequences, the phylogenetic tree is usually constructed through maximum likelihood-based algorithms. To improve the accuracy of these methodologies, many parameters like bootstrap methods, correlation coefficient and residue-substitution models are presumably over-ranked to derive biologically credible relationships. Although the accuracy of protein sequence alignment and the substitution matrix are preliminary constraints to define the biological accuracy of the overlapped sequences/residues, the alignment is not iteratively optimized through the statistical testing of residue-substitution models. The study majorly highlights the potential pitfalls that significantly affect the accuracy of an evolutionary protocol. It emphasizes the need for a more accurate scrutiny of the entire phylogenetic methodology. The need of iterative optimizations is illustrated to construct a biologically credible and not mathematically optimal tree for a sequence dataset.

Insights into the Selectivity Mechanisms of Grapevine NIP Aquaporins

Nodulin 26-like intrinsic proteins (NIPs) of the plant aquaporin family majorly facilitate the transport of physiologically relevant solutes. The present study intended to investigate how substrate selectivity in grapevine NIPs is influenced by the aromatic/arginine (ar/R) selectivity filter within the pore and the possible underlying mechanisms. A mutational approach was used to interchange the ar/R residues between grapevine NIPs (VvTnNIP1;1 with VvTnNIP6;1, and VvTnNIP2;1 with VvTnNIP5;1). Their functional characterization by stopped-flow spectroscopy in Saccharomyces cerevisiae revealed that mutations in residues of H2/H5 helices in VvTnNIP1;1 and VvTnNIP6;1 caused a general decline in membrane glycerol permeability but did not impart the expected substrate conductivity in the mutants. This result suggests that ar/R filter substitution could alter the NIP channel activity, but it was not sufficient to interchange their substrate preferences. Further, homology modeling analyses evidenced that variations in the pore radius combined with the differences in the channel’s physicochemical properties (hydrophilicity/hydrophobicity) may drive substrate selectivity. Furthermore, yeast growth assays showed that H5 residue substitution alleviated the sensitivity of VvTnNIP2;1 and VvTnNIP5;1 to As, B, and Se, implying importance of H5 sequence for substrate selection. These results contribute to the knowledge of the overall determinants of substrate selectivity in NIPs.

Single Residue Substitution at N-Terminal Affects Temperature Stability and Activity of L2 Lipase

Rational design is widely employed in protein engineering to tailor wild-type enzymes for industrial applications. The typical target region for mutation is a functional region like the catalytic site to improve stability and activity. However, few have explored the role of other regions which, in principle, have no evident functionality such as the N-terminal region. In this study, stability prediction software was used to identify the critical point in the non-functional N-terminal region of L2 lipase and the effects of the substitution towards temperature stability and activity were determined. The results showed 3 mutant lipases: A8V, A8P and A8E with 29% better thermostability, 4 h increase in half-life and 6.6 °C higher thermal denaturation point, respectively. A8V showed 1.6-fold enhancement in activity compared to wild-type. To conclude, the improvement in temperature stability upon substitution showed that the N-terminal region plays a role in temperature stability and activity of L2 lipase.