The Structural And Functional Studies Of Sod1 And FibN From Bombyx Mori

Silkworm is an important economic insect to produce the silk. We solved the structure of Sodl and the N-terminal domain of fibroin, to figure out how they function base on their structures.(1) Cu/Zn superoxide dismutases (Sodl) are a large family of cytosolic antioxidant proteins involved in responses to oxidative stress. They catalyze the disproportionation of superoxide radicals into less toxic hydrogen peroxide and dioxygen. Mutations in human Cu/Zn-SOD (hSod1) are associated with about 20% of the familial cases of amyotrophic lateral sclerosis (fALS), a fatal neurodegenerative disorder in heterozygotes. Although the mechanism of this toxicity is still unknown, the aberrant aggregation of hSod1 mutants is strongly suggested to play an important role in the etiology of fALS.The Cu/Zn-SOD from the silkworm Bombyx mori (BmSod1) shares a sequence identity of 63% with hSOD1, with 56 different residues out of the total 153 residues and residue T76 which could be mapped to the corresponding ALS-linked hSOD1 mutation sites.Here, we determined the crystal structure of BmSod1 at high resolution. The overall structures of the holo as well as the Zn-BmSod1 preserve the typical Sodl tertiary structure. Crystal packing of both Cu/Zn-BmSod1 and Zn-BmSod1 showed helical fibril arrangement in the crystalline environment via newly gained nonnative interfaces. The nonnative interfaceⅠis also formed by the zinc loop (involving residues P73, S74, S75, and A76) from one dimer packing onto the exposed edge of P6 (involving S98,199, Q100, and S102) from the neighboring subunit, leading to helical fibrils. The buried interface is up to 650A2. The helical fibrils further stick together to form a water-filled nanotube with an outer diameter of 120A and a water-filled inner cavity of 40A in diameter. The nonnative interfaceⅡbetween helical fibrils buried up to 450A2 on average, involving interactions between the electrostaticloop residues E132-L133 and zinc loop residues E66-K67. The fibril arrangement of Cu/Zn-BmSod1 and Zn-BmSodl in the crystalline environment suggests an ALS-linked mutant-like nature of the wild-type BmSod1. The nonnative interface involved in fibril assembly is located in the electrostatic loop, zinc loop, and strandβ6, all of which are suggested as major candidates for forming aggregation-prone interfaces. We speculated that Sodl might be an intrinsically aggregation-prone protein during earlier stages of phylogenesis, but underwent substantial site mutations opposing self-aggregation during evolution because endogenous protein aggregates are highly toxic and even lethal to mammalian/human neurons.(2) One of the most remarkable biomaterials silk from Bombyx mori, secreted by silk glands (SG) which is a pair organ present in the larval stadium, consists of two types of major proteins, fibroin and a serial sericin. Fibroin heavy chain, as the main core composition of fibroin presents a special organization, with low-complexity crystalline domains made up of Gly-X repeats covering 94% of the sequence and non-repetitive N- and C-terminal of fibroin heavy chain (FibN and FibC). Under various conditions such as pH, concentration, shear stress, dehydration and metal ion changed to minic the silk spinning process, low-complexity crystalline sequence was responsible for the structural transition of fibroin from an initial random coil to an orderedβ-sheet structure implying the extraordinary mechanical properties of the silk. However, there is little study about FibN and FibC. FibN and FibC are hydrophilic properties for promoting the solubility of micelles, which is an important form at the onset of fibroin assembly prior to a liquid crystal phase.In this work, we report that the crytsal structure of FibN 126 is a homodimer of an eight-strandedβ-sheet, in which four strands contributed by a monomer are interwinned with each other. The FibN126 undergoes a structural transition from random coil toβ-sheets when the pH falls to 6.0 or below. This peptide region has intrinsicβ-sheet structures and largely random coil structures in neutral phosphate buffer. Acid environment, hydrophobic interaction and shearing force can all trigger the conformation transition of FibN from random coil toβ-sheet structure. The longer the polypeptide is, the easier the conformational transition undergoes. The serial FibN domain can form the nanoglobular structure with an average diameter ranging from 50 to 100 nm under the condition that the second structure of the proteins changed toβ-sheet. FibN have two distinct roles in the silkgland and during the spinning process. The nanoglobular structure will cover the fibroin heavy chain hydrophobic repetitive sequence and increasing the solubility of the fibroin heavy chain in silkgland. The hydrophilic FibN, firstly to sense the environmental transformation, act as fibroin folding promoter at the initiation stage of the spinning process. Ultimately, the P sheet structure of FibN will be folded into one part of fibrils structure.