Structure of recombinantly expressed cockroach Lili-Mip protein in glycosylated and deglycosylated forms

Dhanabalan, KanagaVijayan; Subramanian, Rudra; Santhakumari, Partha Radhakrishnan; Chavas, Leonard M. G.; Subramanian, Ramaswamy; Banerjee, Sanchari

doi:10.1016/j.bbagen.2021.130064

Cited by 4 publications

(25 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These modified lipocalins are called Anticalins [19]. In tear lipocalin and Lili-Mip 2, Zinc is involved in crystal packing [10,13]. Zinc binds at the entrance of the calyx and is responsible for the preferential binding of Lili-Mip 2 to palmitoleic acid rather than oleic acid [10].…”

Section: Introductionmentioning

confidence: 99%

“…Structures of Lili-Mip and Lili-Mip2 provide a molecular basis for fatty acid binding [8,10]. The Glutamate(E38) at position 38 forms a kink in the ligand binding site in Lili-Mip2 (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

“…The copyright holder for this preprint this version posted December 21, 2022. ; https://doi.org/10.1101/2022.12.21.521413 doi: bioRxiv preprint Structures of Lili-Mip and Lili-Mip2 provide a molecular basis for fatty acid binding [8,10]. The Glutamate(E38) at position 38 forms a kink in the ligand binding site in Lili-Mip2 (Figure 1).…”

mentioning

confidence: 99%

“…It is held in position by histidine (H115) and tyrosine (Y40). One could hypothesize that it favors the binding of Lili-Mip2 to omega-6 unsaturated fatty acids [10]. In tear lipocalin, charged residues Glutamate 34, Histidine 84, and Lysine 114 are thought to be playing a role in ligand interaction [13].…”

mentioning

confidence: 99%

“…This has been observed for another lipocalin-like retinol-binding protein, RBP4 [22], that binds laurate, palmitate, oleate, and linoleate. Glutamate 38 and Phenylalanine at 98 and 100 are conserved between these structures [8,10,22,23]. However, the carboxylate group faces inwards and interacts with tyrosine and arginine in the proteins of the fatty acid binding proteins (FABPs) family [24].…”

mentioning

confidence: 99%

See 4 more Smart Citations

Variability of Phenylalanine side chain conformations facilitates promiscuity of Fatty acid binding in Cockroach milk proteins

Santhakumari

Dhanabalan

Virani

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

The pacific beetle cockroach, Diploptera punctata, is a viviparous cockroach that produces a milk-like substance to support the growing embryo with a brood sac. The structure of the in vivo grown crystals present in the gut of the embryo showed that the milk-derived crystals are heterogenous and are made of three proteins (called Lili-Mips). Multiple fatty acids could be modeled into the active site, and we hypothesized that each of the three isoforms of the protein bound to a different fatty acid. We previously reported that the recombinantly expressed Lili-Mip2 has a structure similar to the structure of the protein determined from in vivo crystals, and this single isoform also binds to several fatty acids. In this study, we aimed to probe the specificity and affinity of fatty acid binding and test the stability of different isoforms. We show that all the isoforms can bind to different fatty acids with very similar affinities, and the local abundance of a fatty acid determined bound fatty acid ratios. Lili-Mips' thermostability is pH dependent, where stability is highest at acidic pH and declines as the pH increases to physiological levels near 7.0. The measurement of the pH in the gut lumen and the gut cells suggests that the pH in the gut is acidic and the pH inside the gut cells is closer to neutral pH. We propose that the protein has evolved to be highly stable in the acidic gut lumen and, when absorbed inside the gut cells, becomes less stable to enable the breakdown of the glycosylated lipo-protein complex to provide essential metabolites for survival and development of the embryo. The different orientations of Phe-98 and Phe-100 control the binding pocket volume and allow the binding of different chain-length fatty acids to bind with similar affinities.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Variability of Phenylalanine side chain conformations facilitates promiscuity of Fatty acid binding in Cockroach milk proteins

Santhakumari

Dhanabalan

Virani

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Glycans modulate lipid binding in Lili-Mip lipocalin protein: insights from molecular simulations and protein network analyses

SureshKumar,

Appadurai,

Srivastava

2023

Glycobiology

View full text Add to dashboard Cite

The unique viviparous Pacific Beetle cockroaches provide nutrition to their embryo by secreting milk proteins Lili-Mip, a lipid-binding glycoprotein that crystallises in-vivo. The resolved in-vivo crystal structure of variably glycosylated Lili-Mip shows a classical Lipocalin fold with an eight-stranded antiparallel beta-barrel enclosing a fatty acid. The availability of physiologically unaltered glycoprotein structure makes Lili-Mip a very attractive model system to investigate the role of glycans on protein structure, dynamics, and function. Towards that end, we have employed all-atom molecular dynamics simulations on various glycosylated stages of a bound and free Lili-Mip protein and characterised the impact of glycans and the bound lipid on the dynamics of this glycoconjugate. Our work provides important molecular-level mechanistic insights into the role of glycans in the nutrient storage function of the Lili-Mip protein. Our analyses show that the glycans stabilise spatially proximal residues and regulate the low amplitude opening motions of the residues at the entrance of the binding pocket. Glycans also preserve the native orientation and conformational flexibility of the ligand. However, we find that either deglycosylation or glycosylation with high-mannose and paucimannose on the core glycans, which better mimic the natural insect glycosylation state, significantly affects the conformation and dynamics. A simple but effective distance- and correlation-based network analysis of the protein also reveals the key residues regulating the barrel’s architecture and ligand binding characteristics in response to glycosylation.

show abstract

Glycans modulate lipid binding in Lili-Mip lipocalin protein

Kumar

Appadurai

Srivastava

2023

Preprint

View full text Add to dashboard Cite

The unique viviparous Pacific Beetle cockroaches provide nutrition to their embryo by secreting milk proteins Lili-Mip, which is a lipid-binding glycoprotein that crystallizes in vivo. The resolved in vivo crystal structure of variably glycosylated Lili-Mip shows a classical Lipocalin fold with an eight-stranded antiparallel beta-barrel enclosing a fatty acid. The availability of physiologically unaltered glycoprotein structure makes Lili-Mip a very attractive model system to investigate the role of glycans on protein structure, dynamics, and function. Towards that end, we have employed all-atom molecular dynamics simulations on various glycosylated stages of a bound and free Lili-Mip protein and characterized the impact of glycans and the bound lipid on the dynamics of this glycoconjugate. Our work provides important molecular-level mechanistic insights into the role of glycans in the nutrient storage function of the Lili-Mip protein. Our analyses show that the glycans locally stabilize spatially proximal residues and regulate the low amplitude opening motions of the residues at the entrance of the binding pocket. Glycans, which are located at the portal end of the barrel, also restrict the distal barrel depth and allosterically modulate the lipid dynamics in the barrel. A simple but effective distance-based network analysis of the protein also reveals the role of glycans in the subtle rewiring of residues crucial for determining the barrel depth and lipid orientation.

show abstract

Structure of recombinantly expressed cockroach Lili-Mip protein in glycosylated and deglycosylated forms

Cited by 4 publications

References 31 publications

Variability of Phenylalanine side chain conformations facilitates promiscuity of Fatty acid binding in Cockroach milk proteins

Variability of Phenylalanine side chain conformations facilitates promiscuity of Fatty acid binding in Cockroach milk proteins

Glycans modulate lipid binding in Lili-Mip lipocalin protein: insights from molecular simulations and protein network analyses

Glycans modulate lipid binding in Lili-Mip lipocalin protein

Contact Info

Product

Resources

About