| Neuronal PER-ARNT-SIM protein 4(NPAS4),one member of the bHLH-PAS transcription factor family,is mainly distributed in the brain and is involved in regulating the expression of a series of downstream genes related to brain cognition and memory.It plays a major role in maintaining the excitation-inhibition balance of neural circuits and regulating cognitive and social functions of the brain.In addition to nerve cells,NPAS4 is also present in islets and vascular epithelial cells.NPAS4 dysfunctions is associated with neurodevelopmental disorders such as depression,schizophrenia,and autism.Therefore,it can be considered as a potential new target for the treatment of these neuropsychiatric disorders,as well as a potential drug target for type 2 diabetes,certain diabetes complications and cardiovascular diseases.bHLH-PAS transcription factors can be divided into two categories:Class Ⅰproteins(NPAS1,NPAS2,NPAS3,NPAS4,HIF-α,SIM1,SIM2,AHR,AHRR and CLOCK)must form a heterodimer with Class II proteins(ARNT,ARNT2,BMAL1 and BMAL2)to activate the expression of downstream genes.Notably,NPAS4 and SIM1 in Class I can form heterodimers not only with ARNT but also with ARNT2,and then regulate the expression of downstream genes by recognizing and binding to specific DNA sequences on downstream target genes.Early research results showed that the dimerization pattern of NPAS4 with ARNT or ARNT2 might be very different from the previously solved dimeric structures of this family,such as HIF-α-ARNT,NPAS1-ARNT,and NPAS3-ARNT-DNA complexes.However,there has been no report about the structure of NPAS4 heterodimers.Therefore,crystal structure of NPAS4 heterodimers(with ARNT or ARNT2)will provide direct evidence to help understand how NPAS4 forms heterodimers with ARNT(or ARNT2),improve people’s understanding of the physiological function of NPAS4,and further reveal the mechanism of how different bHLH-PAS proteins recognize their dimerization partners.In this study,the crystal structures of NPAS4-ARNT2-DNA and NPAS4-ARNTDNA complexes were obtained by X-ray diffraction for the first time.Furthermore,we discovered that NPAS4 subunit has a unique folding pattern compared with HIF-α(including three isoforms HIF-1α,HIF-2α and HIF-3α),NPAS1 and NPAS3 subunits.In addition,the dimerization patterns of NPAS4-ARNT and NPAS4-ARNT2 are not exactly the same.Besides,a structure of protein complex containing the ARNT2 subunit has been solved for the first time.Simultaneously,by a comparative analysis with structures of HIF-α-ARNT,NPAS1-ARNT and NPAS3-ARNT,the new dimerization pattern between NPAS4 and ARNT expands our understanding on ARNT,which is an important dimerization partner for the bHLH-PAS transcription factors.By solving the NPAS4-ARNT/ARNT2 heterodimeric structures(each containing bHLH,PAS-A and PAS-B domains),we found that,there are direct interactions between bHLH and PAS-A domains,as well as between PAS-A and PAS-B domains in the NPAS4 subunit.As a whole,these three domains form a very tight structure.Compared with the structures of HIF-α,NPAS1 and NPAS3 subunits,the β-sheet face of the NPAS4 PAS-B domain is mainly oriented towards its own PAS-A domain,which results in a larger interaction surface between these two domains of NPAS4.At the same time,there is an extra-long α-helix(Jα,16 amino acids)at the end of the PAS-B domain of NPAS4.The insertion of this Jα helix between the PAS-A and PAS-B domains of NPAS4,helps to enhance the interactions between these two domains.The PAS-B domain of CLOCK subunit also possesses an a helix at the same position.However,this helix has a much shorter length(8 amino acids)and takes a different orientation.Meanwhile,at the end of PAS-B domains of HIF-α,NPAS1 and NPAS3 subunits,there are a long loop region and an a helix(6 amino acids)that is much shorter than the Jαhelix of NPAS4.This short a helix is inserted between its own PAS-B domain and the ARNT PAS-B domain during the dimerization process,enhancing the interactions between two subunits.In the PAS-B domains of previously solved Class I members,Gβ strand breaks into two discontinuous short β strands(G1β and G2β)in HIF-α,NPAS1,NPAS3 and CLOCK subunits.On the contrary,Gβ in NPAS4 PAS-B domain shows as a completeβ strand,likely due to the insertion of a single amino acid(M282:methionine).In the NPAS4-ARNT2-DNA and NPAS4-ARNT-DNA complexes,although the amino acid sequence of NPAS4 subunit is completely identical,the overall conformation of NPAS4 subunit in NPAS4-ARNT2-DNA is a little different from that in the NPAS4-ARNT-DNA complex.The results indicated that the conformation of NPAS4 subunit may change when paired with different dimerization partners(ARNT2 or ARNT),to adapt to different dimerization objects.In these two heterodimeric complexes,ARNT2 and ARNT subunits are wrapping around the outside of their paired NPAS4 subunits,similar to the previously solved heterodimeric ARNT subunits.Meanwhile,the bHLH,PAS-A and PAS-B domains of ARNT and ARNT2 subunits,interact with their paired NPAS4 subunit to form three discrete divisions.On the other hand,there is a big difference in the overall conformation between the ARNT2 subunit and ARNT subunit during the dimerization process with NPAS4.The sequence identity between ARNT2 and ARNT in bHLH,PAS-A and PAS-B domains is 91%,78%and 79%,respectively.When directly superimposing each of the bHLH,PAS-A and PAS-B domains,the RMSD value of their Cα is 1.0 ?,0.72 ? and 0.80 A,respectively.However,the spatial arrangement of the three domains between ARNT2 and ARNT is actually very different with a RMSD value of 3.2 A for all the Cα together.The ARNT2 PAS-A domain has an outward deflection of about 20° as compared with the ARNT PAS-A domain.At the same time,the relative positions of PAS-B domains in ARNT and ARNT2 proteins are also different.The conformational differences between NPAS4,ARNT and ARNT2 in each complex lead to differences in interaction junctions between individual domains within the heterodimers of NPAS4-ARNT and NPAS4-ARNT2.By comparing the ARNT subunits in different heterodimeric structures of this family,we found that although ARNT’s sequences are exactly the same,including the three identical domains(bHLH,PAS-A and PAS-B),there are obvious differences in the overall arrangement of these domains.From the comparison results,it can be seen that ARNT subunits paired with different dimerization partners have great differences in structure.On one hand,it is likely caused by the flexibility of the arrangement of ARNT’s discrete domains;on the other hand,the structural differences of different dimerization partners may lead to the varied arrangement of ARNT’s three domains in the dimerization process.In conclusion,in this study we analyzed the complex structures of NPAS4-ARNT2 and NPAS4-ARNT,and revealed the unique folding pattern of NPAS4,which is a new pattern different from those of the other Class I members of the bHLH-PAS family.The dimerization pattern of NPAS4 subunit is slightly different between ARNT and ARNT2.Compared with HIF-α-ARNT,NPAS1-ARNT and NPAS3-ARNT heterodimers,we found that NPAS4-ARNT showed a novel dimerization pattern.The discovery of the new interaction mode lays the structural foundation for further revealing the adaptation of ARNT to different dimerization partners.It also provides structural information for potentially regulating the transcriptional activity of NPAS4 complexes and for developing drugs against NPAS4-related diseases. |
