• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Diffusion of nucleotide excision repair protein XPA along DNA by coarse-grained molecular simulations?

    2021-10-28 07:18:44WeiweiZhang張偉偉andJianZhang張建
    Chinese Physics B 2021年10期
    關鍵詞:張建

    Weiwei Zhang(張偉偉) and Jian Zhang(張建)

    National Laboratory of Solid State Microstructures,School of Physics,Collaborative Innovation Center of Advanced Microstructures,Nanjing University,Nanjing 210093,China

    Keywords: nucleotide excision repair,XPA,one-dimensional diffusion along DNA,molecular simulation

    1. Introduction

    Nucleotide excision repair(NER)is the central DNA repair pathway widely observed in eubacteria, eukaryotes and archaea. It is responsible for the removal of DNA lesions caused by UV irradiation,chemical mutagens,and other stress damages.[1–5]The abnormalities of NER can lead to the xeroderma pigmentosum(XP)disease,which is featured by sensitivity to sunlight and high risk of developing skin cancers.[6,7]XPA (xeroderma pigmentosum complementation group A) is a key protein in NER,and mutation of XPA gene can lead to severe disease symptoms.[6,8]It has been showed that the XPA may play multiple roles in NER.[9–12]For example,in addition to preferentially binding to various types of DNA lesions and distorted substrates,[10,13]XPA has also been found to interact with several NER related proteins during the damage recognition and verification steps,acting as a scaffold.[11,14]

    The human XPA has the length of 273 amino acids and is composed of three domains, including the intrinsically disordered N-terminal region(N-region),the central DNA-binding domain(DBD),and the intrinsically disordered C-terminal region (C-region) (see Fig. 1). The flexibility of the two disordered regions were suggested to promote the binding of XPA to several partners,[15–19]while the central globular DNAbinding domain was found to bind with DNA and interact with numerous NER proteins as well.[20]

    Due to its biological importance in NER, XPA has received extensive attention.[7,21]Although previous investigations mostly focused on the interactions of the XPA at the DNA lesion site during the damage recognition and verification steps, recent experimental study showed that detecting the DNA lesions may also involve the one-dimensional diffusion of the XPA at the non-damage site of DNA.[22]Therefore,more detailed investigations for the molecular dynamics of the XPA diffusion along the non-damage site of DNA are important for deeper understanding the biological function of XPA in the NER.

    Although all-atom detailed molecular dynamics (MD)simulation has been widely used to directly simulate the details of the inter-molecular interactions involved in the protein-DNA recognitions, it is still extremely timeconsuming to study the whole diffusion process of proteins along DNA.[23–26]Recently, the combination of the atomic-interaction-based coarse-grained model(AICG2+)for proteins[27,28]and the three-site-per-nucleotide (3SPN.2C)model for DNA[29]has shown great success in studying the dynamical and structural features of the protein-DNA interactions.[30–33]Here,based on these models,we performed extensive coarse-grained molecular simulations to study the diffusion dynamics of XPA along the DNA.From our simulations,we found that XPA diffuses along DNA with the DNAbinding residues similar to that identified from crystal structure. It was also found that the diffusion of XPA along DNA is achieved through combination of one-dimensional and threedimensional mechanisms. At low salt concentrations,the onedimensional diffusion with strong rotational coupling is the dominant mechanism. At higher salt concentrations, the diffusion by three-dimensional mechanism becomes more probable. Meanwhile,the one-dimensional diffusion of XPA along DNA displays sub-diffusive behaviour at wide range of salt concentrations. In addition, the simulation showed that the XPA becomes more extended in the presence of DNA or at high salt concentration,suggesting that both DNA binding and increasing salt concentration may promote the binding of other target proteins to XPA by increasing the exposure extent of the binding sites.

    2. Model and method

    2.1. Coarse-grained model of protein-DNA system.

    Throughout this work, to model protein (XPA), the structure-based coarse-grained model was used, where each amino acid in protein was coarse-grained to a single bead located at Cαposition. The interactions between protein beads include AICG2+ potentials and Debye–H¨uckel-type electrostatic interactions. The AICG2+ energy function is given byVAICG2+=Vlocal+VGo+Vexv, whereVlocal,VGO, andVexvare local potentials, non-local structure-based potentials, and excluded volume potentials respectively. The details of these terms can be found in Refs. [28,34,35], and the similar energy function has also been successfully used in the studies of protein folding,enzyme catalysis,chromatin remodeler and nucleosome dynamics in recent works.[27,36–40]Note that the structure-based interactions were omitted for the disordered regions in XPA, and effects of salt concentration are implicitly modeled in the Debye–H¨uckel-type interactions with the details displayed as follows:

    whereλDis the Debye length,ε(T,C) is the dielectric constant,andIis ionic strength.

    To model dsDNA,the 3SPN.2C model,[29]developed by de Pablo’s group, was used. In this model each nucleotide is simplified to three CG particles, representing the phosphate group (P), sugar (S), and base (B). The interactions among these particles are composed of structure-based local potentials,base pairing,intrastrand base-stacking,interstrand crossstacking, excluded volume effects, and Debye–H¨uckel-type electrostatic interactions. The partial charge of the phosphate beads was set as?0.6e to consider the effect of local counterion condensation effect.[31,41]The 3SPN.2C model model is able to capture several properties of dsDNA and has been widely used to study the DNA-protein interactions.

    For the protein-DNA interactions,only the excluded volume effects and the electrostatic interactions were considered. As the electrostatic interaction plays a dominant role in protein-DNA interactions, we paid special attention to this term in the study of protein-DNA binding. For the structured motif in proteins, the partial charges of the CG beads were parametrized by the RESPAC method,[42]which can provide more accurate description of the electrostatic potential. In describing the electrostatic interactions between the protein and DNA,the partial charge of the phosphate bead was set as?1.0,following the work in Ref.[31].

    2.2. Simulation details

    The model for XPA was built based on the available crystal structure(PDB entry:6j44[8])with missing residues added by modeller9.23.[43]The sequence and structure of XPA are shown in Figs. 1(a), 1(b), and 1(c). For DNA, two random sequences with different lengths were prepared: R100, R150(the sequences and the structures are shown in Figs.1(d)and 1(e)). In our work, the reference structures for DNA were built with the 3DNA package.[44]For the protein-DNA system, the initial structures for DNA and protein in all the simulations were separated and placed randomly. During production runs,the center of mass(COM)of DNA was restrained to the original position and COM distance between XPA(DBD)and DNA was restrained to be smaller than 250 °A.The ionic strength was set to 50, 150, 250, 350, and 450 mM. At each ionic strength,24 simulations were carried out independently,each lasting for 5×108MD steps for XPA-R100 system and over 4×108MD steps for XPA-R150 system,respectively. To characterize the structure features of XPA in the absence of DNA,we also performed several simulations with cumulative length of approximately 1×109MD steps. For all the simulations,we recorded the coordinates of XPA and DNA at every 1×104MD steps. The first 2×107frames were omitted in the calculations of distributions during the posterior analysis.

    All the CG simulations were performed by the CafeMol3.2 package.[34]The simulations were conducted by Langevin dynamics with friction coefficientγ=0.02 at temperatureTof 300 K.The structures were visualized by using pymol.[45]

    2.3. Analysis

    To characterize the search modes of XPA on DNA, we defined a DNA recognition motif consisting of the residues 140, 141, 142, 151, 179, 207, 211,[46]which forms contacts with DNA in the crystal structure. Following the approach used in Ref. [41], the diffusion is considered as sliding if at least five of these residues maintain staying within 15 °A from the DNA.Correspondingly,the diffusion is considered as three-dimensional diffusion if all of the seven residues in the recognition motif leaves the DNA surface by at least 25 °A.In comparison,the protein is considered to perform the hopping dynamics if these residues are 15 °A–25 °A away from DNA.Based on above criteria, we classified both sliding and hopping as one-dimensional diffusion mechanism following the work in Ref[41].

    To describe the location of DBD searching site on DNA during one-dimensional diffusion,we used the mean position of the nucleotides that closest to the recognition motif(RM):

    In the above definition,tis simulation time,Lis 3.4 °A (one bp along the strand), RM is the recognition motif, composed of the residues 140,141,142,151,179,207,and 211,NRMis the number of residues in recognition motif,bp(j)is the base pair index of particlejin DNA;δkj=1,ifjis the DNA bead closest to the protein beadk;otherwiseδkj=0.[31]

    The protein bead and DNA bead form a contact if their distance is less than 7 °A.Additionally,we defined several local coordinate axes, following the procedure in Ref. [47], to characterize the rotational and translational motions of XPA around DNA.

    Fig.1. Sequence and structure of the XPA and DNA studied in this work. (a)Domain architecture of XPA.(b)The sequence of XPA protein with each domain shadowed by the same color as in panel(a). The secondary structure features of DBD domain is also shown. (c)The available crystal sturcture of DBD is shown in cartoon representation and the color scheme for secondary structures is the same as in panel(b).The disorderd regions are represented by dotted lines with the same color as in panel(a). The key DNA-binding residues(residue 140,141,142,151,179,207,211)are shown in ball-and-stick representation and colored differently. The sequences(d)and structures(e)of DNA in this study.

    3. Results

    3.1. XPA binding and diffusion along DNA

    Firstly, to quantify the structural properties of nonspecific interaction interface between XPA and DNA, based on two random DNA sequences with different lengths(R100,R150), we performed MD simulations for the XPA binding to the nonspecific dsDNA site at various salt concentrations and calculated the probabilities of contact formation between the DNA and each residue of the XPA at various salt concentrations. As shown in Fig. 2, there is a striking similarity in the landscape of contact probability between R100 and R150,indicating the conserved binding interface of XPA regardless of properties of DNA.Additionally,for both R100 and R150,one can see that, besides the DBD, residues in the N-region also have high contacting probabilities with DNA,which suggests that the N-region can be involved in the XPA–DNA interactions during the searching process of damage sites. Meanwhile, the results clearly show that increasing salt concentrations tends to decrease the contact probabilities, but without altering the peak locations of the contact probability distributions, which may suggest that the XPA uses the same set of residues for DNA binding at wide range of salt concentrations.Particularly, the residues with high contact probabilities have large overlap with the DNA-binding residues observed in crystal structure of the XPA–DNA complex,[46]demonstrating that the DNA binding surface in the DBD is conserved in the specific and non-specific binding. Such behaviour of maintaining binding interface was also observed in other DNA-binding proteins,[47]and was suggested to facilitate the search of the cognate site on DNA.[48,49]

    Fig. 2. Contact probabilities of the XPA residues with DNA at various salt concentrations. Residues in the DBD domain are shaded by pink backgound.The key DNA-binding residues in the crystal structure are highlighted by magenta circles. The result with R100(R150)is shown in the upper(lower)panel and a representative structure with DBD bound to R100 is also displayed.

    Fig.3. (a)and(b)Time series of the distance between the COM of N-region and DNA surface(green)and that between the COM of DBD and DNA surface (red) at 250 mM with sequence R100 (a) and R150 (b). When calculating the distance between the COM of N-terminal and DNA surface, only the residues ranging from 30 to 50, which have high probabilities of forming contacts with DNA, were considered. (c) and (d) Probabilities of onedimensional diffusion(green)and three-dimensional diffusion(pink)mechanism at different salt concentrations with sequence R100(c)and R150(d).

    Besides the properties of binding interface,we also characterized the binding and diffusion process of XPA along DNA. Considering the relatively higher probabilities of contact formation with DNA for the residues in the DBD and the N-region, we plotted the time series of the distances between the above two protein segments and the DNA surface to illustrate the binding dynamics. The centers of mass(COM)of the DBD and that of the residues 30–50 were used to represent the positions of the DBD and the N-region relative to DNA surface. From Figs.3(a)and 3(b), one can clearly see the attachment and detachment processes of the two XPA segments.Such attachment and detachment processes are consistent with experimental observation.[22]However, it is worthy to note that,because of the limited accessible time in our simulation,the repetition of attachment and detachment is only observed sporadically at relative high salt concentration (Fig. 3(a)).Additionally,inspection of trajectory of these two protein segments demonstrated that the attachment/detachment of the DBD and N-region are correlated, lacking of the anchoring and binding mechanism mediated by disordered region as typically observed in other DNA-binding proteins containing disordered regions.[50]After the simultaneous detachments of the N-region and the DBD,the whole XPA performs threedimensional diffusion and then rebinds to the DNA(Fig.3(a)).For many DNA binding proteins, rapidly finding their specific binding sites on DNA in response to external stimulus is vitally important. Thus, in addition to the random threedimensional diffusion driven by thermal diffusion,the protein also uses other searching mechanisms, such as sliding,[51–53]hopping or intersegmental transfer.[54]During sliding,the protein can tightly bind to DNA and diffuse along the DNA helix using the recognition region,[55]in which electrostatic interaction between protein and DNA plays dominant role. Whereas in the hopping diffusion process,the electrostatic interactions are much weaker, which allows the protein to diffuse more freely along the DNA surface and accelerates diffusion by skipping several DNA bases.[41]Unlike three-dimensional diffusion, in which the protein can diffuse far away from DNA,the protein remain staying close to the DNA during the sliding and hopping,which tends to significantly reduce the conformational space during target site search and therefore accelerate the searching of the target site in the DNA.[51]Previous studies showed that the combination of these different search modes is needed to achieve the maximal target-search efficiency.[54,56]Therefore, we also analyzed the probabilities of the one-dimensional diffusion mechanism and threedimensional diffusion mechanism (criteria about classifying different mechanisms can be found in Model and Method) at various salt concentrations for sequence R100 and R150, respectively (Figs. 3(c) and 3(d)). From these results, one can see that, increasing salt concentration can decrease the probability of one-dimensional searching mechanism and increase the three-dimensional searching mechanism for each DNA sequence. As shown in the Debye–Hc¨ukel model, the effect of salt concentration is mainly achieved by changing the length of Debye radius. With the increasing of salt concentrations,the screening effect of the implicit salt ions increases, leading to the decrease of electrostatic interactions. Such results again suggest the dominate role of electrostatic interaction in the XPA–DNA binding.

    Considering the important role of one-dimensional diffusion, we then analyzed the diffusion kinetics and calculated the mean square displacement(MSD)of the one-dimensional DBD translational motions along the DNA (MSD(?t) =〈(X(t+?t)?X(t))2〉, where〈〉represents the time average)as a function of time difference ?tat various salt concentrations, and results are shown in Fig.4. The fitting by a power law function MSD(?t) =K?tαwere also shown in the inset by log–log scale. From our results, one can clearly see that the MSD of XPA (DBD) increases upon increasing salt concentration during one-dimensional diffusion for both R100 and R150. The fitting of the diffusion kinetics by the above power law function for R100 and R150 both give theα<1.0,which suggests sub-diffusive motion of DBD along DNA(inset figure in Fig. 4) at wide range of salt concentrations and may facilitate the search of target DNA sequences as suggested by other works.[57–59]Furthermore,this kind of sub-diffusion may be related to the diverse strengths of DNA–XPA interactions,which may be ascribed to the variations of the DNA conformations and the fluctuations of the DNA-protein binding.This kind of connection is consistent with those in many other sub-diffusive dynamics,[59–69]and can be described with continuous time random walk(CTRW)model.[61,69]Note that the exponents of the sub-diffusive dynamics are almost invariant at different salt concentrations for each sequence. This observation suggests that the distributions of the diverse strengths are insensitive to the variations of salt concentrations, since the conformational fluctuations are intrinsic for the the DNAprotein system.This result bring us more insights on the DNAprotein interactions.

    Fig.4. Mean square displacement(MSD)of the one-dimensional XPA diffusion along the DNA(R100: upper panel,R150: lower panel)as a function of simulation time at different salt concentrations.The results of power function fitting α are also shown in the inset.

    Fig.5. Translational–rotational coupling in the one-dimensional diffusion of XPA along DNA.(a)Representative trajectory fragments of rotation(θ) and translation (y) of XPA along R100 (R150) at the salt concentrations of 50 mM, 150 mM, and 450 mM are shown in upper panel (lower panel). Representive structures with DBD bound to DNA at 50 mM and 450 mM are displayed. (b)The correlation plots between ?θ and ?y of XPA at 50 mM,150 mM,and 450 mM(R100: upper panel,R150: lower panel).

    For DNA-interacting proteins, several studies have suggested that diffusion along the DNA helical path is required to achieve efficient target search.[55,70]Meanwhile,the rotationcoupled sliding was proposed to be the general mechanism for the one-dimensional search of protein on DNA.[71]Therefore,we also investigated whether the one-dimensional diffusion of XPA is along helical pitch(rotation-coupled)or not(rotationuncoupled), and the results are shown in Fig. 5. To more appropriately describe this dynamical process,we defined several local axes following the Ref.[47]. The coordinates of the seven residues in the DNA-binding motif mentioned above were used to calculate the rotational angle and translational position along the DNA helical axis. In Fig. 5(a), we plotted the time series of rotational and translational motions of DBD along the R100 and R150 at various salt concentrations during the one-dimensional diffusion. From these trajectories, one can see that the protein can diffuse randomly along DNA at these different salt concentrations. At relatively low salt concentrations(50 mM,150 mM),where the electrostatic interactions are strong, the one-dimensional diffusion occurs dominantly by the rotation-coupled mechanism. While at high salt concentration(450 mM),the electrostatic interactions become weaker, which makes the protein move more freely along the DNA axis, leading to weak correlation between translational and rotational motions. To more clearly characterize the relationship between rotation and translation during one-dimensional diffusion, we also showed the rationaltranslational correlation plot in Fig.5(b)for all trajectories at three different salt concentrations. From these results,one can see an obvious correlation between the rotation and translation at low salt concentrations (50 mM), while it disappears at relatively high salt concentration (450 mM). Particularly,the rotational–translational correlation is still significant at the physiological relevant salt concentration(150 mM).

    3.2. Structural properties of XPA

    In addition to the diffusion features during this dynamical process, the structural properties of XPA were also studied,since its structure dynamics will surely affect the binding of XPA with other binding partners. The results for these two different DNA sequences are shown in Fig. 6. From our results, one can clearly see that both the DNA binding and varying salt concentration can change the structural properties of XPA and the difference in the effects of R100 and R150 on protein structure at each salt concentration is negligible.In detail, as shown in Fig. 6(a), at low salt concentration(50 mM), due to the strong intra-molecule electrostatic interactions in XPA, these two disordered regions pack against the DBD, leading to small radius of gyration (Rg) of XPA(less than 30 °A) in the absence of DNA, but it increases a lot (around 40 °A) upon binding DNA, which may be caused by the occupancy of intra-molecule interacting sites by DNA.While at high salt concentration (450 mM), the electrostatic interactions between intra and inter-molecule is decreased significantly and consequently the intra and inter molecule motions are more freely, leading to largeRgof XPA with or without DNA, and the effects of DNA binding is much deprecated as well (Fig. 6(b)). What is more, in Figs. 6(c)and 6(d), we also plotted the distribution of contact number between DBD and the disordered regions. In accordance with the above analysis, there are a large number of contacts between N(C)-terminal and DBD,and it decreases much unpon binding DNA at low salt concentration (Fig. 6(c)),while at high salt concentration,the contact number decreases significantly with or without DNA.Notably,the contact number in the absence of DNA is still less compared with binding DNA.From the analysis of the contact number,it becomes clear that both increasing salt concentrations and DNA binding can lead to the decrease of contact number,indicating more extended conformation of XPA. Based these observations, considering the dominate role of electrostatic interactions in describing the XPA–DNA interactioins and N(C)–DBD interactions in XPA, we can conclude that, both the DNA and salt concentration can act as modulating factors of the XPA structure.

    Fig.6. Effect of DNA binding and salt concentration on the conformation of XPA.(a)and(b)Rg distribution of XPA with(R100: blue,R150:purple)and without(black)DNA binding at the salt concentrations of 50 mM(a)and 450 mM(b). The representative structures of XPA are also shown,and the color scheme for domains is the same as in Fig.1(a). (c)and(d)Distribution of contact number formed between the DBD domain and the two disordered regions of XPA with(R100: blue,R150: purple)and without(black)DNA binding at the salt concentrations of 50 mM(c)and 450 mM(d).

    Nowdays, more and more studies have found that the conformation of protein is very sensitive to external stimulus, which play an important role in modulating the conformation of protein involed in various biological activities, like a cascade of reactions or signal transduction in cells,etc.[72–76]Interestingly, previously studies have also found that both salt concentration and binding DNA could affect the protein structure.[77–82]Hence,toghter with the findings that the XPA needs to cooperate with other binding partners during the NER pathway, and multiple binding sites for these partners are located at both DBD and disordered regions(N-terminal and Cterminal),[16,17,20,83–86]we can speculate that both the DNA itself and the salt concentration play a role in modulating the NER and at high salt concentration,the protein predominately adopt an extended conformation, ready for interacting with other partners,while at relative low salt concentration,the disordered regions tend to pack tightly with the DBD without DNA, however, upon DNA binding, the N(C)–DBD contacts are much reduced,which can lead to increased exposure of the binding sites in XPA for recruiting other partner proteins.

    4. Conclusion

    Here, we used a coarse-grained model to study the dynamical process of XPA diffusion along two different DNAs.Our results showed that the diffusion properties of XPA along these two DNAs are pretty similar,which indicates the robustness of dynamical behaviour of XPA along DNA.From more detailed perspective,our results showed that the XPA diffuses along the DNA with interface similar to the recognition motif found in the crystal structure, which may promote the search efficiency for the DNA target. Additionally, we also characterized the diffusion process of XPA along DNA. The XPA diffuses along DNA using combination of one-dimensional and three-dimensional search mechanisms,and increasing salt concentration can decrease the proportion of one-dimensional mechanism. During one-dimensional diffusion, the protein displays subdiffusive motions, which has also been observed in several biological system. Additionally, the correlation analysis of rotational and translational motions of DBD along DNA also showed that at relatively low salt concentration,the one-dimensional DBD translational motion is coupled to rotation,that is to say,sliding along helical pitch. Whereas at high salt concentration,the correlation disappears. All these results suggest that, like many other DNA binding proteins,[54,87,88]XPA maintains its position and orientation with respect to targeted DNA base pairs during one-dimensional diffusion at relatively low salt concentration, ensuring the efficient target search.

    In addition to binding to DNA, XPA has been found to act a key scaffold protein, interacting with many proteins involved in NER.[86]Therefore,we analyzed the structural features of XPA at various salt concentrations,in the presence and absence of DNA, respectively. The results showed that both DNA and salt concentration can modulate the protein structure. At low concentration,DNA binding can significantly increase theRgof protein and decrease the contact number between DBD and disordered regions,indicating more extended conformation of XPA.While at high concentration, the weak interaction between protein and DNA and intra-molecular interaction will let the protein to be in the extensible state as well. Based on these discussions, we can speculate that even at relatively low salt concentrations,when XPA bind to DNA,the contacts between DBD and other disordered regions are reduced, which tends to increase the exposure extent of the binding sites for other repair proteins,promoting the scaffold function of the XPA.

    Acknowledgment

    The authors thank the insightful discussions with Jun Wang,Wenfei Li,and Wei Wang.

    猜你喜歡
    張建
    鷸蚌相爭,漁人得利
    張建一:匠人匠心 征服世界的聲音
    綠色中國(2019年13期)2019-11-26 07:10:58
    本期焦點人物:張建一
    綠色中國(2019年13期)2019-11-26 07:10:50
    河鲀投毒案
    Les gar?ons sont-ils privilégiés?
    法語學習(2016年5期)2016-12-18 15:16:23
    那還用說
    那還用說
    那還用說
    愛你(2015年11期)2015-11-17 10:42:55
    那還用說
    肩關節(jié)生物力學
    x7x7x7水蜜桃| 亚洲精品在线美女| xxx96com| 老司机深夜福利视频在线观看| 搡老熟女国产l中国老女人| 看片在线看免费视频| 亚洲中文日韩欧美视频| 99精品在免费线老司机午夜| 精品国内亚洲2022精品成人| 一个人看视频在线观看www免费 | 黄色女人牲交| 少妇人妻一区二区三区视频| 国产免费一级a男人的天堂| 日韩欧美在线乱码| 欧美日韩国产亚洲二区| 男女午夜视频在线观看| 91麻豆精品激情在线观看国产| 一二三四社区在线视频社区8| 夜夜爽天天搞| 蜜桃久久精品国产亚洲av| 国产亚洲精品av在线| 两性午夜刺激爽爽歪歪视频在线观看| 人人妻人人澡欧美一区二区| 中文字幕熟女人妻在线| 精品熟女少妇八av免费久了| 成人欧美大片| 免费在线观看影片大全网站| 长腿黑丝高跟| av女优亚洲男人天堂| 成人18禁在线播放| 在线十欧美十亚洲十日本专区| 日韩人妻高清精品专区| 一个人免费在线观看的高清视频| 成年免费大片在线观看| 成人永久免费在线观看视频| 欧美av亚洲av综合av国产av| 岛国在线免费视频观看| 成人亚洲精品av一区二区| 亚洲无线观看免费| 美女大奶头视频| 亚洲性夜色夜夜综合| 国产成人aa在线观看| 久久久精品大字幕| 我的老师免费观看完整版| 午夜免费观看网址| 亚洲五月天丁香| 又爽又黄无遮挡网站| 一边摸一边抽搐一进一小说| 99国产精品一区二区三区| 岛国在线观看网站| 日韩国内少妇激情av| 在线a可以看的网站| 每晚都被弄得嗷嗷叫到高潮| 日本五十路高清| 国产伦精品一区二区三区视频9 | 91在线精品国自产拍蜜月 | 波野结衣二区三区在线 | 床上黄色一级片| 国内毛片毛片毛片毛片毛片| 午夜免费激情av| 亚洲av免费高清在线观看| 狂野欧美白嫩少妇大欣赏| 日日摸夜夜添夜夜添小说| 国产99白浆流出| 色吧在线观看| 九色成人免费人妻av| 久久午夜亚洲精品久久| 欧美高清成人免费视频www| 日韩人妻高清精品专区| 精品免费久久久久久久清纯| 国产v大片淫在线免费观看| 久久精品国产亚洲av涩爱 | 亚洲内射少妇av| 成人av在线播放网站| 国产激情欧美一区二区| 不卡一级毛片| 99在线人妻在线中文字幕| 最近在线观看免费完整版| 国产综合懂色| 日本黄色视频三级网站网址| 久久久久久国产a免费观看| 无人区码免费观看不卡| 在线国产一区二区在线| 男女午夜视频在线观看| 亚洲七黄色美女视频| 国产精品久久电影中文字幕| 午夜两性在线视频| 国产精品三级大全| 夜夜夜夜夜久久久久| 两性午夜刺激爽爽歪歪视频在线观看| 久久亚洲精品不卡| 欧美成狂野欧美在线观看| 中文字幕久久专区| 亚洲 国产 在线| 香蕉av资源在线| 在线观看66精品国产| 国内精品久久久久精免费| 少妇的逼水好多| 手机成人av网站| 国产三级中文精品| 久久久久久久久大av| 亚洲av一区综合| 禁无遮挡网站| 国产真实乱freesex| 欧美丝袜亚洲另类 | 久久草成人影院| 国内精品一区二区在线观看| 亚洲18禁久久av| 操出白浆在线播放| 成人高潮视频无遮挡免费网站| 国产不卡一卡二| 亚洲精华国产精华精| 中文在线观看免费www的网站| 三级国产精品欧美在线观看| 中文字幕熟女人妻在线| 成人18禁在线播放| 麻豆久久精品国产亚洲av| 麻豆国产97在线/欧美| 国内精品久久久久久久电影| 国产毛片a区久久久久| 亚洲美女视频黄频| 午夜精品久久久久久毛片777| 精品久久久久久久人妻蜜臀av| 人人妻人人澡欧美一区二区| 亚洲精品久久国产高清桃花| 脱女人内裤的视频| 午夜福利18| 成人午夜高清在线视频| 国产又黄又爽又无遮挡在线| 欧美一区二区精品小视频在线| 中文字幕熟女人妻在线| 国产精品 欧美亚洲| 一区二区三区激情视频| 成人特级av手机在线观看| xxxwww97欧美| 精品福利观看| 九色成人免费人妻av| 桃色一区二区三区在线观看| 日本成人三级电影网站| 琪琪午夜伦伦电影理论片6080| av片东京热男人的天堂| 全区人妻精品视频| 久久精品综合一区二区三区| 成熟少妇高潮喷水视频| 国产爱豆传媒在线观看| 亚洲黑人精品在线| 国产在线精品亚洲第一网站| 在线观看av片永久免费下载| 91久久精品国产一区二区成人 | 国产不卡一卡二| 俺也久久电影网| 国产av麻豆久久久久久久| 亚洲乱码一区二区免费版| 母亲3免费完整高清在线观看| 国产成人av激情在线播放| 中文字幕av成人在线电影| 精品免费久久久久久久清纯| 看免费av毛片| 亚洲美女黄片视频| 黄片大片在线免费观看| 欧美日韩国产亚洲二区| 美女高潮喷水抽搐中文字幕| 免费看日本二区| 九九在线视频观看精品| 天天添夜夜摸| 老师上课跳d突然被开到最大视频 久久午夜综合久久蜜桃 | 国产男靠女视频免费网站| а√天堂www在线а√下载| 国产综合懂色| www日本黄色视频网| 十八禁人妻一区二区| 成人欧美大片| 色综合亚洲欧美另类图片| 久9热在线精品视频| 久久久久免费精品人妻一区二区| 美女黄网站色视频| 首页视频小说图片口味搜索| 1000部很黄的大片| 午夜亚洲福利在线播放| 啦啦啦韩国在线观看视频| 精品久久久久久,| 国产成人影院久久av| 波多野结衣巨乳人妻| 97人妻精品一区二区三区麻豆| 桃红色精品国产亚洲av| 亚洲最大成人中文| 国产一区二区激情短视频| 欧美一级毛片孕妇| 夜夜看夜夜爽夜夜摸| 亚洲国产欧美人成| 99久久成人亚洲精品观看| 日本免费一区二区三区高清不卡| 天天躁日日操中文字幕| 亚洲国产日韩欧美精品在线观看 | 中文字幕久久专区| avwww免费| 欧美色视频一区免费| 日本熟妇午夜| 国产精品爽爽va在线观看网站| 熟女少妇亚洲综合色aaa.| 不卡一级毛片| 一夜夜www| 日韩av在线大香蕉| 一级毛片女人18水好多| 亚洲成人中文字幕在线播放| 女警被强在线播放| 国产精品久久久久久久电影 | 亚洲精品一卡2卡三卡4卡5卡| 成年女人永久免费观看视频| 一卡2卡三卡四卡精品乱码亚洲| 国产一级毛片七仙女欲春2| 18禁在线播放成人免费| 啦啦啦观看免费观看视频高清| 欧美av亚洲av综合av国产av| 真人一进一出gif抽搐免费| 国产精品99久久久久久久久| 麻豆国产97在线/欧美| 亚洲男人的天堂狠狠| 久久久久久九九精品二区国产| 69av精品久久久久久| 窝窝影院91人妻| 亚洲片人在线观看| 精品一区二区三区视频在线 | 久久午夜亚洲精品久久| 757午夜福利合集在线观看| 九色成人免费人妻av| 国产精品久久电影中文字幕| 久久人人精品亚洲av| a在线观看视频网站| 搡老熟女国产l中国老女人| 亚洲,欧美精品.| 99久久精品国产亚洲精品| 九九在线视频观看精品| 国产精品久久久久久人妻精品电影| 国产精品电影一区二区三区| 亚洲,欧美精品.| 久久草成人影院| 欧美一区二区精品小视频在线| 欧美成人a在线观看| 中文字幕高清在线视频| 国产精品久久久人人做人人爽| 亚洲人成伊人成综合网2020| 一进一出抽搐gif免费好疼| 日韩av在线大香蕉| 欧美绝顶高潮抽搐喷水| 中文字幕熟女人妻在线| 国产欧美日韩精品一区二区| 婷婷六月久久综合丁香| 午夜福利成人在线免费观看| 久久人人精品亚洲av| 最近在线观看免费完整版| 国产精品国产高清国产av| 国产精品日韩av在线免费观看| svipshipincom国产片| 欧美又色又爽又黄视频| 亚洲国产色片| 麻豆成人av在线观看| 欧美日韩乱码在线| 免费av观看视频| 97超视频在线观看视频| 级片在线观看| 欧美在线黄色| 欧美中文日本在线观看视频| 一区二区三区高清视频在线| 国产精华一区二区三区| 国产亚洲精品久久久久久毛片| 日本a在线网址| 看黄色毛片网站| 国产精品一及| 日本三级黄在线观看| 日本五十路高清| 麻豆国产av国片精品| 成年版毛片免费区| 欧美性猛交黑人性爽| 制服人妻中文乱码| 国产精品久久久久久精品电影| 色视频www国产| 每晚都被弄得嗷嗷叫到高潮| 国产亚洲精品久久久com| 老熟妇乱子伦视频在线观看| av视频在线观看入口| 国产亚洲精品综合一区在线观看| 床上黄色一级片| 欧洲精品卡2卡3卡4卡5卡区| 精华霜和精华液先用哪个| 黄色片一级片一级黄色片| 中文亚洲av片在线观看爽| 成人午夜高清在线视频| 在线观看舔阴道视频| 波多野结衣高清作品| 99热这里只有精品一区| 丁香欧美五月| aaaaa片日本免费| 每晚都被弄得嗷嗷叫到高潮| 男人和女人高潮做爰伦理| 精品福利观看| 久久亚洲真实| 成年版毛片免费区| 欧美成人性av电影在线观看| 欧美+日韩+精品| 久久精品综合一区二区三区| 美女被艹到高潮喷水动态| 97超视频在线观看视频| 一本精品99久久精品77| 国产蜜桃级精品一区二区三区| 99久国产av精品| 日韩精品青青久久久久久| 久久九九热精品免费| 一个人看的www免费观看视频| 超碰av人人做人人爽久久 | 亚洲,欧美精品.| 国内久久婷婷六月综合欲色啪| 午夜福利18| 一级黄片播放器| 每晚都被弄得嗷嗷叫到高潮| 午夜精品久久久久久毛片777| 欧美日韩综合久久久久久 | 免费av毛片视频| 91麻豆av在线| 少妇人妻一区二区三区视频| 99久久综合精品五月天人人| 午夜福利欧美成人| 婷婷丁香在线五月| 午夜福利18| aaaaa片日本免费| 好男人电影高清在线观看| 亚洲av免费高清在线观看| 欧美区成人在线视频| 国产精品自产拍在线观看55亚洲| 午夜福利成人在线免费观看| 国产精品嫩草影院av在线观看 | 亚洲成人免费电影在线观看| 国产一区二区在线av高清观看| 两人在一起打扑克的视频| 欧美日韩综合久久久久久 | 啦啦啦韩国在线观看视频| 亚洲成人中文字幕在线播放| 欧美乱妇无乱码| 国产精品亚洲美女久久久| 18禁国产床啪视频网站| 无人区码免费观看不卡| 精品福利观看| 成人特级av手机在线观看| 国产久久久一区二区三区| 久久久久久人人人人人| 亚洲精华国产精华精| 亚洲国产欧美网| 18禁国产床啪视频网站| xxxwww97欧美| 亚洲成人免费电影在线观看| 亚洲内射少妇av| 久久国产精品人妻蜜桃| 最好的美女福利视频网| 亚洲国产欧美人成| 无人区码免费观看不卡| 91九色精品人成在线观看| 国产一区二区在线观看日韩 | 夜夜躁狠狠躁天天躁| 亚洲av成人不卡在线观看播放网| 国产精品精品国产色婷婷| 国产精品亚洲av一区麻豆| 91九色精品人成在线观看| 国产私拍福利视频在线观看| 免费在线观看亚洲国产| av天堂中文字幕网| 噜噜噜噜噜久久久久久91| 亚洲国产精品久久男人天堂| 又粗又爽又猛毛片免费看| 一个人观看的视频www高清免费观看| 夜夜躁狠狠躁天天躁| 3wmmmm亚洲av在线观看| 一级作爱视频免费观看| 老司机在亚洲福利影院| 丰满乱子伦码专区| 色尼玛亚洲综合影院| 伊人久久大香线蕉亚洲五| 一边摸一边抽搐一进一小说| 亚洲精品在线美女| 午夜精品在线福利| 国内少妇人妻偷人精品xxx网站| 国产淫片久久久久久久久 | 国产三级在线视频| 性欧美人与动物交配| 日本与韩国留学比较| 性欧美人与动物交配| 亚洲精品成人久久久久久| 久久草成人影院| 3wmmmm亚洲av在线观看| 黄色丝袜av网址大全| 一区福利在线观看| 尤物成人国产欧美一区二区三区| 亚洲av美国av| 日日摸夜夜添夜夜添小说| 90打野战视频偷拍视频| 一个人看视频在线观看www免费 | 国内少妇人妻偷人精品xxx网站| 一进一出抽搐动态| 日本与韩国留学比较| 免费看日本二区| 久久精品国产自在天天线| 亚洲不卡免费看| 久久久成人免费电影| 天堂影院成人在线观看| 国产av不卡久久| 国产色婷婷99| 久久久精品欧美日韩精品| 欧美+日韩+精品| 成人午夜高清在线视频| 欧美区成人在线视频| tocl精华| 嫩草影视91久久| 在线看三级毛片| 国产精品影院久久| www国产在线视频色| 国产一区二区在线观看日韩 | 丁香欧美五月| 欧美国产日韩亚洲一区| 久久久久久久久久黄片| 99久久精品一区二区三区| 久久久久久大精品| 亚洲最大成人中文| 性色avwww在线观看| 身体一侧抽搐| x7x7x7水蜜桃| 一a级毛片在线观看| www.熟女人妻精品国产| 国产成年人精品一区二区| 国产伦精品一区二区三区四那| 亚洲av日韩精品久久久久久密| 搡老熟女国产l中国老女人| 69人妻影院| 老司机在亚洲福利影院| 亚洲av免费高清在线观看| 日本免费一区二区三区高清不卡| 女人被狂操c到高潮| 久久亚洲精品不卡| 国产成人av激情在线播放| 成年女人永久免费观看视频| 欧美日韩国产亚洲二区| 91久久精品国产一区二区成人 | 我要搜黄色片| 变态另类成人亚洲欧美熟女| 嫁个100分男人电影在线观看| 成人特级黄色片久久久久久久| 久久性视频一级片| 精品人妻偷拍中文字幕| 中出人妻视频一区二区| 欧美午夜高清在线| 国产av麻豆久久久久久久| 国产精品女同一区二区软件 | 人人妻人人看人人澡| 偷拍熟女少妇极品色| 一本综合久久免费| 日日干狠狠操夜夜爽| 国产老妇女一区| 久久久色成人| 午夜福利18| 日本三级黄在线观看| 免费电影在线观看免费观看| 国产在线精品亚洲第一网站| 国产一区二区三区在线臀色熟女| 丰满乱子伦码专区| 91在线观看av| 啪啪无遮挡十八禁网站| 不卡一级毛片| 国产亚洲欧美98| 精品国产三级普通话版| 中文字幕久久专区| 久久99热这里只频精品6学生| 欧美最新免费一区二区三区| 能在线免费观看的黄片| av国产免费在线观看| av黄色大香蕉| 精品一区在线观看国产| 成年版毛片免费区| 国产淫片久久久久久久久| 自拍偷自拍亚洲精品老妇| 高清毛片免费看| 天堂√8在线中文| 国产伦一二天堂av在线观看| 欧美xxxx黑人xx丫x性爽| 中文天堂在线官网| 国产v大片淫在线免费观看| 七月丁香在线播放| 国产成人午夜福利电影在线观看| 69av精品久久久久久| 欧美xxⅹ黑人| 女人被狂操c到高潮| 美女国产视频在线观看| 看黄色毛片网站| 91久久精品国产一区二区成人| 人体艺术视频欧美日本| 三级毛片av免费| 午夜日本视频在线| 韩国av在线不卡| 日日啪夜夜撸| 精品亚洲乱码少妇综合久久| 免费不卡的大黄色大毛片视频在线观看 | 日韩视频在线欧美| 18禁在线播放成人免费| 美女脱内裤让男人舔精品视频| 最近的中文字幕免费完整| 男人舔女人下体高潮全视频| 欧美97在线视频| 深爱激情五月婷婷| 三级男女做爰猛烈吃奶摸视频| 国产精品久久久久久精品电影小说 | 成人综合一区亚洲| 精品国产三级普通话版| 国产v大片淫在线免费观看| 少妇被粗大猛烈的视频| 亚洲精品国产av蜜桃| 成人国产麻豆网| 国产在线一区二区三区精| 五月伊人婷婷丁香| 蜜桃久久精品国产亚洲av| 国产精品久久久久久精品电影| 又爽又黄a免费视频| 国产 亚洲一区二区三区 | 亚洲美女搞黄在线观看| 3wmmmm亚洲av在线观看| 亚洲人成网站在线播| 精品久久久久久电影网| 中文在线观看免费www的网站| xxx大片免费视频| 真实男女啪啪啪动态图| 亚洲自偷自拍三级| 午夜精品一区二区三区免费看| 激情 狠狠 欧美| 看十八女毛片水多多多| 97精品久久久久久久久久精品| 国产精品av视频在线免费观看| 可以在线观看毛片的网站| 69人妻影院| 成人毛片60女人毛片免费| 特大巨黑吊av在线直播| 国产成人福利小说| 国产白丝娇喘喷水9色精品| 亚洲国产精品成人久久小说| 男女下面进入的视频免费午夜| 日韩电影二区| 国产成人a∨麻豆精品| 91午夜精品亚洲一区二区三区| 别揉我奶头 嗯啊视频| 国产亚洲最大av| 三级男女做爰猛烈吃奶摸视频| 亚洲精品成人av观看孕妇| 看非洲黑人一级黄片| 联通29元200g的流量卡| 少妇猛男粗大的猛烈进出视频 | 亚洲av免费在线观看| av在线亚洲专区| av国产久精品久网站免费入址| 蜜臀久久99精品久久宅男| 男人狂女人下面高潮的视频| 免费电影在线观看免费观看| 国产伦理片在线播放av一区| 99久国产av精品国产电影| 美女主播在线视频| 99久久精品一区二区三区| 亚洲精品影视一区二区三区av| 亚洲av国产av综合av卡| 国产 一区 欧美 日韩| 18禁在线播放成人免费| 寂寞人妻少妇视频99o| 成人亚洲精品av一区二区| 色综合站精品国产| 最近中文字幕2019免费版| 日本黄色片子视频| 亚洲av成人av| 一个人看的www免费观看视频| 久久人人爽人人片av| 免费电影在线观看免费观看| 久久精品国产亚洲av涩爱| 亚洲国产精品成人久久小说| 草草在线视频免费看| 欧美日本视频| 日本欧美国产在线视频| 男人舔女人下体高潮全视频| 久久久欧美国产精品| 日本一二三区视频观看| 日韩中字成人| 久久久精品免费免费高清| 美女内射精品一级片tv| 国产精品福利在线免费观看| 一级爰片在线观看| 免费观看无遮挡的男女| 亚洲精品乱码久久久久久按摩| 国产综合懂色| 国精品久久久久久国模美| 国产高潮美女av| 伊人久久国产一区二区| 国产精品爽爽va在线观看网站| 夫妻性生交免费视频一级片| 女人被狂操c到高潮| 精品久久国产蜜桃| 最近中文字幕2019免费版| 亚洲精品456在线播放app| 一级a做视频免费观看| 精品国产露脸久久av麻豆 | 男人舔奶头视频| 国产 一区 欧美 日韩| 欧美激情在线99| 国产永久视频网站| 中文字幕人妻熟人妻熟丝袜美| 日本爱情动作片www.在线观看| 中文字幕av成人在线电影| 国产 一区 欧美 日韩| 女人十人毛片免费观看3o分钟| 亚洲激情五月婷婷啪啪| av黄色大香蕉| 国产黄片视频在线免费观看| 在线观看av片永久免费下载| 中文在线观看免费www的网站| 一区二区三区四区激情视频|