The Crystal Structures And Functional Analysis Of Proteorhodopsin

Microbial rhodopsins are a kind of photoactive membrane chromoproteins,sharing a common architecture of seven transmembrane a-helices(helix A-G).Retinal chromophore is covalently attached by a protonated Schiff base linkage to a conserved lysine in the seventh helix(helix G),which is employed to absorb photons for light-energy conversion or the initiation of intracellular signaling.In the last 15 years,proteorhodopsins(PR)have been widely distributed in bacteria,viruses,fungi and archaea from all over the world,such as marine,freshwater,sea ice,high mountains or even Siberian permafrost samples.In fact,PRs comprise the most abundant members of the microbial rhodopsin superfamily,While sharing practically less than 30%amino-acid sequence identity,proteorhodopsin(mainly eubacterium)and bacteriorhodopsin(BR)from Halobacterium salinarum,functions as a similar light-activated proton pumps that transport protons from the cytoplasmic to the extracellular side of the cells with using light.The parts of functional residues in the ion transfer pathway are similar between PR and bR.Asp85 and Asp96 of BR are conserved in PR(Asp97 and Glu105)at an equivalent postition,as primary proton acceptor and donor of the Schiff-base.By contrast,the proton release group(BR Arg82,Glul94 and Glu204)lack a pair of glutamic acids in PR(Arg94),suggesting that the ion-transfer mechanism may differ between PR and BR.However,to date very little structural information is available,especially crystal structure,and so many questions remain unclear.HOT75BPR is a blue-absorbing proteorhodopsin(BPR)gene isolated from the Pacific Ocean near Hawaii at a depth of 75 m.Crystals of the HOT75BPR D97N mutant were obtained as reported previously,but the structure is not resolved with the limit of resolution.In this study,based on the crystallization conditions,the recombinant protein expressed in E.Coli is purified using immobilized metal affinity chromatography(IMAC)and is used to crystallize by vapor diffusion method.We successfully get good quality native crystals and the selenomethionine crystals are prepared using the same condition.HOT75BPR D97N crystal structure is resolved by Se-SAD method.This is the first crystal structure of a blue light absorption proteorhodopsin and its diffraction is 2.7A.HOT75BPR D97N crystal is belong to space group P21212.Each PR monomer is comprised seven transmembrane a-helices(helix A-G)and retinal chromophore is located between the seven helices.There are some remarkable differences between the HOT75BPR D97N crystal structures and other reported structures of microbial rhodopsins.The loop region between helices B and C is much shorter.Instead of the anti-parallel β-sheet that is observed between helix B and C in other microbial rhosopsins,only four residues Gly87-Pro90 form a short β-turn.This region near the proton-release group has a cavity and direct access to the extracellular side.Helix E is connected to via a helical distortion at Gly196 and helix G contains a kink at residue Asn231 similar to the π-bulge.These features maybe affect the proton transfer.We find that proton transfer mechanism is different in PR and BR.A new and conserved glutamic acid,Glu142,in the D-E loop,is involved in the release of protons and coordinated via hydrogen bonds to three neighboring tyrosine Tyr95,Tyr208 and Tyr224.The glutamate is connected to Arg94,and together help proton release into the extracellular.Wat402,a key molecule responsible for proton translocation between the Schiff base and the proton acceptor in BR,appears to be absent in PR.These features are also observed in another BPR(from the Mediterranean Sea at a depth of 12 m,Med12BPR)crystal structure solved by us.Previous results showed another distinct difference between PR and BR was the pKa of the Schiff-base proton acceptor.The pKa of Asp97 in PR is much higher(7-8)than the Archaea BR(less than 3.0).HOT75BPR crystal structure shows a C5 pentamer and five peptide chains together have interactions.Comparison of PR and BR,the interface regions involve different helices,especially hydrogen bonds and salt bridges in the N-terminal and helix A.The most striking feature of proteorhodopsin is the position of the conserved histidine throughout PRs,which close to the conserved Asp97 from the same molecule and forms a hydrogen bond to Trp34 of a neighboring protomer.This is a novel intermolecular interaction without other microbial rhosopsins.All PR sequence alignment and Med12BPR structure analysis suggest that this hydrogen bonding may be conserved in the BPR.In addition,the carboxylate group of Asp22 in the N-terminal region forms intermolecular hydrogen bonds to the residues Thr91,Val92 and Phe93 located on helix C of the neighboring protomer.The previous mutation of Asp22 could cause the decrease of the pKa of Asp97.To reveal the mechanism behind the pKa modulation,we solve the crystal structure of D22V.Compared to HOT75BPR D97N crystal structures,this phenomenon may be related to the state of the oligomer.The mutant of Asp22 results in the disruption of the intermolecular interactions and the change of hydrogen-bond network in Trp34-His75-Asp97.Therefore,we propose a regulation mechanism on the pKa of Asp97 that inter-protomer interactions of Trp34 and Asp22 modulate the pKa of Schiff-base counterion Asp97 through His75.PRs found in marine microbes are the most abundant retinal-based photoreceptors on this planet.PR variantsa(blue light-absorbing proteorhodopsin,BPR,and a green light-absorbing proteorhodopsin,GPR)show high levels of environmental adaptation,as their colors are tuned to the optimal wavelength of available light.The two major green and blue subfamilies(GPR:λmax = 525 nn;BPR:Amax = 490nm)can be interconverted through a L/Q point mutation at position 105.Here we successfully investigate the absorption properties of 20 amino acid residues into position 105 of BPR.Absorption peaks show all types contain all-trans retinal as predominant,and three mutants(Q105K,Q105W and Q105Y)obviously have another retinal isomers.The observed absorption properties reveal the λmax values of all the mutants occur red-shift and there is a positive correlation to some extent.between the volume of residues at 105 position side chain and absorption spectra.Via the optimization of crystallization conditions for the wild-type and 19 mutant proteins of position 105 in HOT75BPR,we successfully resolve four crystal structures,respectively wt(λmax = 496 nm),105C(λmax = 521 nm),105M(λmax = 533 nm)and 105F(λmax = 543 nm).Absorption spectra of these cover blue,green and maximum redshift wavelengths.Here we reveal the structural basis behind this intriguing color-tuning effect at 105 position.Three factors are normally considered:(ⅰ)retinal structural distortion,(ⅱ)the distance between Schiff base and counterion,(ⅲ)electrostatic interaction and steric in retinal polyene chains around the charged,polarity and aromatic amino acids.