Structural Biology Research On Human PABPN1 RRM

(â… )In most eukaryotes,poly(A)tails are attached to the 3'-ends of mRNAs.They influence almost all aspects of mRNA metabolism:transport from nuclear to cytoplasm,translation and turnover.To mediate their biological functions,poly(A) tails are covered in sequence-specific manner by two types of conserved poly(A) binding proteins,PABPC in the cytoplasm and PABPN1 in the nucleus.PABPC(Pablp in Saccharomyces cerevisiae)is present in all eukaryotes,It plays important roles in the initiation of translation and in mRNA decay.PABPC contains four copies of RRMs(the RNA recognition motif,also known as RBD(RNA-binding domain))in its N-terminal portion.RRM1 and RRM2 are most similar to RRM3 and RRM4,respectively.The first two RRMs are mainly responsible for specific binding to poly(A).In the crystal structure of an human PABPC(RRM1/2)bound to poly(A), theβ-sheet surfaces form a platform that binds an extended conformation of the oligonucleotide.Each RRM interacts with about four nucleotides.PABPN1(also named PABP2,PABâ…¡)is the only nuclear poly(A)binding protein identified in mammalian so far.It can improve synthesis of the poly(A)tails by stimulation of PAP(poly(A)polymerase)and control poly(A)tail length.Under native conditions,there was equilibrium between monomers,dimmers and higher oligomers of the protein.Electron microscopy of PABPN1-poly(A)complexes suggests that oligomerization of the protein is functionally significant.The complexes can form spherical particles which are in equilibrium with filamentous complexes. This dynamic structure may function as a molecular ruler to determine the length of poly(A)tail.Although PABPN1 binds poly(A)with high affinity and specificity as PABPC,it just contains a high conserved single RRM,which separates the argine-rich C-terminal domain from the acidic N-terminal domain which is essential for the stimulation of PAP.Both argine-rich domain and the RRM are required for poly(A)binding,but the latter contributes the high specificity.Intriguingly,despite those sequences which are identified as RRM family,the RRM of PABPN1 has little sequence similarity with any of the RRMs of known structure,including the RRMs of PABPC. Here,crystals or the single RRM or human PABPN1 in two space groups have grown from the same conditions,unusually,in the same droplet.We determined the three-dimensional structures of the two crystals independently.The first structure was determined by single wavelength anomalous diffraction(SAD)method at 2.0(?) resolution with R factor=17.4%(R_free20.7%)in the cubic crystal space group I23, with unit cell dimensions of a(?)b(?)c(?)92.35(?).The second structure was determined by molecular replacement at a resolution of 2.82(?)with R lactor=22.6%(R_free 29.0%)in the trigonal space group P3₁,with unit cell dimensions of a= b=59.34(?), c=80.60(?).The overall structure is very similar in both crystal forms,adopting a fold similar to the canonical RRM topology.However,two apparent features of PABPN1 RRM are distinguished from other RRMs:the loop3 deviation and the observed dimerization in the crystal structure.(â…¡)The formation of 5-Aminolevulinic acid(ALA)is the first committed universal precursor in the tetrapyrrole biosynthesis pathway.In contrast to animal. plants,algae,bacteria and many other bacteria synthesize ALA from glutamate by a C₅ pathway in which the carbon skeleton of glutamate is converted into ALA by a series of enzymes.Glutamatel-semialdehyde am inotransfcrase(GSA-AT),a vitamin B6-dependent enzyme,is the last enzyme in this pathway.It catalyses the net transfer of the C2 amino group of GSA to the C1 position.Here we reported the three-dimensional structure of GSA-AT from Bacillus subtilis, The structure has been determined by X-ray crystallography using molecular replacement,refined at 2.3(?)resolution to an R-factor 19.3%(R_free23.4%).The GSA-AT fold can be divided into three domains.The N-terminal domain consists of about 65 residues that form twoα-helix followed by a three-stranded antiparallelβ-sheet.The cofactor binding domain,residues 65 320,contains a central seven strandedβ-sheet with six parallel strands and one antiparallel strand.The C-terminal domain,comprising residues 321-430,folds into two three-stranded antiparallel β-sheet and four helices,including one at the C terminus.The structure shows the active site loop(residues 145-178,the entrance to the active site)is well ordered,we also present the location and conformation of the cofactor PMP.