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Predicting Weakly Stable Regions, Oligomerization State, and Protein–Protein Interfaces in Transmembrane Domains of Outer Membrane Proteins

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Predicting Weakly Stable Regions, Oligomerization State, and Protein–Protein Interfaces in Transmembrane Domains of Outer Membrane Proteins Predicting weakly stable regions, oligomerization state, and protein–protein interfaces in transmembrane domains of outer membrane proteins Hammad Naveed, Ronald Jackups Jr., and Jie Liang1 Department of Bioengineering, University of Illinois, Chicago, IL 60607 Edited by William F. DeGrado, University of Pennsylvania School of Medicine, Philadelphia, PA, and approved June 11, 2009 (received for review February 26, 2009) Although the structures of many ␤-barrel membrane proteins are membrane proteins, such as voltage-sensing (11), flux control of available, our knowledge of the principles that govern their ener- metabolites, and ion-sensing (9). getics and oligomerization states is incomplete. Here we describe Computational studies have also contributed much to the a computational method to study the transmembrane (TM) do- understanding of ␤-barrel membrane proteins, including the mains of ␤-barrel membrane proteins. Our method is based on a identification of ␤-barrel membrane proteins from sequences of physical interaction model, a simplified conformational space for many microbial genomes, the prediction of their topological efficient enumeration, and an empirical potential function from a orientations, and the characterization of their structural features detailed combinatorial analysis. Using this method, we can identify (12, 13). In addition, numerous spatial and sequence motifs and weakly stable regions in the TM domain, which are found to be ensemble properties of the TM domains have also been studied important structural determinants for ␤-barrel membrane pro- (13–18). As models and algorithms improve, it is natural to ask teins. By calculating the melting temperatures of the TM strands, whether computational studies can reveal further insight on the our method can also assess the stability of ␤-barrel membrane structural organization of ␤-barrel membrane proteins. proteins. Predictions on membrane enzyme PagP are consistent In this study, we explore whether an empirical energy function with recent experimental NMR and mutant studies. We have also and a minimalistic structural model of ␤-barrel membrane BIOPHYSICS AND discovered that out-clamps, in-plugs, and oligomerization are 3 proteins (15, 17) can reveal the structural organizing principles COMPUTATIONAL BIOLOGY general mechanisms for stabilizing weakly stable TM regions. In of non-barrel elements in the TM domain. We also study whether addition, we have found that extended and contiguous weakly the oligomerization state of the TM domains can be predicted stable regions often signal the existence of an oligomer and that and whether there exist unstable or weakly stable regions in TM strands located in the interfaces of protein–protein interactions are strands, despite the existence of extensive H-bond networks. considerably less stable. Based on these observations, we can First, we describe a computational approach for identifying predict oligomerization states and can identify the interfaces of unstable or weakly stable regions of ␤-barrel membrane proteins. ␤ protein–protein interactions for -barrel membrane proteins by We define such regions as TM strands with energy by an empirical using either structure or sequence information. In a set of 25 potential function significantly higher than expectation. Using the nonhomologous proteins with known structures, our method PagP enzyme for which NMR and mutant data are available, we successfully predicted whether a protein forms a monomer or an show that the predicted weakly stable region and the identified key oligomer with 91% accuracy; in addition, our method identified residue contributing to the instability are consistent with experi- with 82% accuracy the protein–protein interaction interfaces by mental observations. We have further identified several general using sequence information only when correct strands are given. strategies where ␤-barrel membrane proteins employ non-barrel elements to stabilize weakly stable regions, including out-clamps in-plug ͉ membrane protein oligomerization ͉ out-clamp ͉ (19), in-plugs, and oligomerization. We then describe the calculated protein–protein interaction ͉ weakly stable TM strand melting temperature, Tm, of the TM domains of the proteins. In addition, we found that there are often extensive and contiguous eveloping a general understanding of how proteins behave weakly stable regions in many ␤-barrel membrane proteins that can ␤ Din membranes is of fundamental importance. -barrel mem- be used to accurately determine whether a membrane protein exists brane proteins, one of the 2 major structural classes of mem- primarily in a monomeric or oligomeric state, and to identify the brane proteins, have been studied extensively. Currently, struc- interface regions for protein–protein interactions in the TM do- ␤ tures of 70 -barrel membrane proteins have been resolved, and main. Our method can be generalized so that oligomerization state much has been learned about their thermodynamic stability (1), and interaction interfaces can be predicted from sequence infor- folding kinetics (2–4), biogenesis (5), and biological functions mation alone, without the knowledge of the three-dimensional (6). These membrane proteins are thought to have very regular structures. structures, with the basic principles of their architectural orga- nization well understood (7). An overwhelming structural fea- Results ture is the existence of an extensive regular hydrogen bond ␤ ␤ The physical model for membrane-embedded -strands is discrete network between the transmembrane (TM) -strands, which is and minimalistic. It assumes neighboring strands register and thought to confer extreme stability on the proteins (8). However, ␤-barrel membrane proteins have diverse structures and often deviate significantly from the standard barrel archi- Author contributions: H.N. and J.L. designed research; H.N. and R.J. performed research; tecture. For example, there are often small ␣-helices and R.J. contributed new reagents/analytic tools; H.N. and J.L. analyzed data; and J.L. wrote the ␤-strands, called in-plugs, found inside the ␤-barrel (9). Non- paper. barrel-embedded helices are also found to pack against the TM The authors declare no conflict of interest. ␤-strands (10). In addition, some ␤-barrel membrane proteins This article is a PNAS Direct Submission. exist in monomeric form, whereas others form oligomers as their 1To whom correspondence should be addressed. E-mail: [email protected]. biological functional unit (9). These structural irregularities and This article contains supporting information online at www.pnas.org/cgi/content/full/ diversity often play important functional roles for ␤-barrel 0902169106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902169106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 28, 2021 2 6 1 4 5 2 3 15 16 789101112 13 14 0 −2 Energy 1 5 Fig. 1. The expected energy values of strands in the 2 3 4 16 TM domain of OmpF. OmpF exists in trimeric form, 6 15 with strands 1–5 and strand 16 forming the interaction Energy 10 11 7 9 13 14 8 12 surface. (Left Inset) The trimeric structure of OmpF. 0123 (Right Inset) The energetic contributions of individual −4 residues. Among the 16 strands, strands 1–6 and −1 strands 15 and 16 are found to have the highest overall 0 50 100 150 200 250 300 350 energy values. These strands coincide with the pro- Strands Residues tein–protein interaction interface. interact through canonical strong H-bonds, weak H-bonds, and ‘‘out-clamp’’ here) to lock the protein in its native form once side-chain interactions. Details of this model and an empirical folding and insertion are complete (19). statistical potential function can be found in refs. 15 and 17 and are Using a full space-filling atomic model of the structure (see SI described in Methods and in supporting information (SI) Appendix. Appendix and ref. 24 for details), we found that this helix interacts predominantly with strands B and D, instead of with Detecting Weakly Stable Regions by Empirical Potential Function. To strands B and C as reported in refs. 10 and 19, in which only the demonstrate the utility of the empirical potential function, we backbone ribbon diagram is considered. Among these, contacts first illustrate the detection of weakly stable regions in the TM with R59, L69, and Y87 account for the majority of the atomic domain of the protein OmpF. OmpF is a homotrimeric TM interactions of barrel strands with the helix protein in which each subunit forms a pore that allows diffusion To assess the extent to which this helix stabilizes the TM of small polar solutes across the outer membrane. After sum- strand, we quantify the stability of the strands using our empir- ming up the energetic contribution of individual TM residues, we ical potential function. Fig. 2 shows the energy contribution at found that the 8 strands with the highest overall energies are different positions of the TM strands in PagP. Our calculation strands 1–6, 15, and 16, and are predicted as the weakly stable indicates that strand B is the most unstable strand and has the strands. It turns out that the strands involved in oligomerization highest energy, with the single-body energy term of residue R59 are strands 1–5 and strand 16 (Fig. 1). Collectively, the weakly contributing most to its instability, despite the modest stabilizing stable strands detected by the empirical potential function form effect of pairwise interactions involving R59. the oligomerization interface (see also Fig. S2 in SI Appendix
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