| Membrane transporters,exist widely in organisms.They can not only mediate the exchange of substances across biological membranes,such as ions,sugars,and amino acids,but also play an important role in signal transduction,energy conversion,mechanosensing,and many other physiological and pathological processes.It is estimated that up to 30%of the human genome codes for membrane proteins,the majority of which are membrane transports,and changes in the structure and function of these proteins often cause transport-related diseases in the human body.In the natural environment,transporters play an important role in the defense of organisms against abiotic stresses such as high salt and heavy metal poisoning and can maintain the stability of osmotic pressure.In industrial microorganisms,transporters control the discharge of intermediate products and final products,which is of great significance for weakening the negative feedback effect of production strains.The analysis of the three-dimensional structure of related proteins is crucial for the elucidation of molecular transport mechanisms,rational modification of proteins,and drug design.Therefore,in recent years,transporters have become a research hotspot in many fields such as biomedicine and industrial microorganisms.The development of protein crystal crystallographic technique,especially cryoelectron microscopy,has greatly enriched the membrane protein structure database.While expanding people’s understanding of the structure and function of transporters,it also provides a basis for the analysis of transporters with unknown structures.The development of protein structure prediction technology such as Alphafold provides a powerful tool for the structure prediction of related proteins.Structural-functional elucidation of proteins from different superfamilies shows that a large number of transporters have widely varying substrate profiles and no significant sequence homology,but they share a similar folding pattern of discontinuous helical structure in the core region with inverted repeat structure.In addition to the classified X-type(NhaA fold)and parallel-type(LeuT fold)discontinuous helices,there are also some transporters with hairpin-type discontinuous helices.It is noteworthy that the presence of amino acid residues in the discontinuous helix sequences,which are closely related to transport and folding,has become a key target for enhancing or inhibiting the activity of related proteins.Due to the characteristics of membrane proteins themselves,technical or equipment limitations,most of the transporters still do not have available structural information.Based on the growing wealth of structural information on transporter and protein structure prediction techniques such as Alphafold,the study of discontinuous helices located in the core region and the resolution of the conservation and characterization of their key structures are important ideas for understanding the structure of related proteins and their molecular transport mechanisms when crystal structures are diffi cult to obtain.(When it is difficult to obtain the structure,based on the increasingly abundant structural information of transporters and protein structure prediction technologies such as Alphafold,studying the discontinuous helix located in the core region and analyzing its characteristics and conservation is the important ideas to understanding the structure of related proteins and their molecular transport mechanisms.)According to the different folding modes of the discontinuous helix,the key Na+/H+ antiporter NhaD in halotolerant bacteria,the major arsenate-antimonate exporter ArsB and Acr3,and the arginine-lysine efflux protein LysE from Corynebacterium glutamicum,were selected for the study of sequence conservation and key structural features analyses of discontinuous helices,respectively.Firstly,we identified their conserved sequences and motifs based on sequence conservation analysis,combined with structural modelling,the folding mode of the discontinuous helices were predicted,and its sequence conservation and characteristics were analyzed.The conserved sites in the core functional areas were further selected for mutagenesis,and the mutants were validated for their physiological function and analyzed for changes in kinetic parameters.Mutant proteins on key residues for substrate binding were constructed and the conformation and substrate affinity changes were studied by fluorescence resonance energy transfer(FRET),trypsin hydrolysis,and Microscale Thermophoresis(MST).The sequence conservation and common structural features of these three discontinuous helices were analyzed and summarized to provide a theoretical basis for the analysis and application development of the molecular transport mechanism of the relevant transport proteins.1.Summarization of the sequence and structural characteristics of IT superfamily proteins with hairpin-type discontinuous helix through the study of NhaD and ArsB.The ion transporter(IT)superfamily contains a variety of Na+/H+ antiporters represented by NhaD and endows organisms with resistance to high-salt environments,but there is a lack of information on their structure and molecular transport mechanisms;while the study of arsenate-antimonate transporters represented by ArsB can provide theoretical basis for the elimination of highly toxic valence arsenic or antimony oxides from the environment.In this study,NhaD and ArsB were selected as representatives to summarize the sequence and structure information of hairpintype discontinuous helix-containing proteins in the IT superfamily.After evolutionary conservation analysis and motif analysis,two important conserved regions and four motifs with similar spatial positions were identified in NhaD and ArsB;among them,Motif A and Motif C are located at the tips of HP1 and HP2,while Motif B and Motif D are located at Loopl and Loop2,the mutation of residues on these conserved motifs resulted in complete loss of protein transport ability.It was confirmed by FRET and trypsin hydrolysis experiments that in NhaD and ArsB,mutations at the sites on Motif A and Motif C(D166,D405;D112,N337)caused significant changes in protein structure.MST experiments of mutants on the four conserved motifs of ArsB showed that some residues in these regions play an important role in protein transport by affecting substrate affinity.In addition,a series of charged residues,e.g.E64,E65,R454,and R464,are predicted to be involved in the net charge switch of NhaD activation,by collectively form an "pH sensor" at the entrance of the cytoplasmic funnel.2.Summarization of the sequence and structural characteristics of X-type discontinuous helix-containing transporters through the study of Acr3Bs.The arsenical resistance-3(ACR3)family constitutes the most common efflux pathway that confers high-leyel resistance to toxic metals in various microorganisms and lower plants.The three-dimensional and topological model of Acr3Bs exhibits a typical NhaA folding pattern,with two discontinuous helices of transmembrane(TM)segments,TM4 and TM9,interacting with each other and forming an X-shaped structure.Three conserved motifs were also identified corresponding to Motif A,B,and C of the hairpin-type discontinuous helical protein,and site-directed mutagenesis.MST,and FRET analyses were used to analyze the important sites of them.The identified Motif C in TM9 was found to be a critical element for substrate binding,in which N292 and E295 are involved in substrate coordination,while R118 in TM4 and E322 in TM10 are responsible for structural stabilization.In addition,the highly conserved residues on Motif B of TM5 are potentially key factors in the protonation/deprotonation process.3.Summarization of the sequence and structural characteristics of the parallel type discontinuous helical tran sporter and the rational modification through the study of LysE.LysE is the only known L-arginine/L-lysine efflux protein in Corynebacterium glutamicum,and the analysis of structural information of this protein could provide an important theoretical basis for the modification of L-arginine and L-lysine producing strains.Taking LysE as an example,the sequence and structural characteristics of parallel discontinuous helices are summarized,and on this basis,rational design and transformation of proteins are carried out to realize the combination of theoretical research and actual production.It is predicted that the:protein consists of six transmembrane helices,among which TM1 and TM4 are discontinuous helices parallel to each other.Four conserved motifs were identified in its two conserved regions and the functions of the amino acids on them were verified.It is speculated that Motif A,B,and C are involved in the transport function of proteins,while Motif R is related to the recognition and screening of substrates,where there are many key sites involved in substrate recognition and binding,such as Q21,D46,N153,and K27.Based on this,the binding sites within 0.4 nm centered on the substrate L-arginine were screened,and the LysE mutant with improved arginine transport ability and decreased lysine efflux activity was obtained by point iterative saturation mutation.4.Common structural and sequence features of three types of discontinuous helix-containing proteins.Analysis and comparison of the sequences and structures of the three discontinuous helical folding patterns revealed that not only does each folding pattern have its own key sequence conservation and common structural features,but there is also structural conservation of the active center structure between them.Three conserved motifs(Motif A,Motif B,Motif C)constituting th e active center and key sites related to substrate binding were identified at the same positions in their threedimensional structures.In addition,these proteins rejy on Na+or H+to drive the process of substrate transport,and there are specific am ino acids in the corresponding proteins that coordinate with the two driving ions,among which acidic amino acids Asp or Glu are essential in H+-driven proteins and Ser residues are involved in the binding of Na+.These amino acid residues are highly conserved in proteins and tend to be located on the three motifs. |
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