The general research aims of my group are to use molecular modeling and bioinformatics to analyze structure, function, and molecular evolution of membrane proteins. Membrane proteins are one of the most important classes of proteins. They comprise about 30% of most genomes and are involved in many biological processes. They are especially important in biomedical research because most targets of current pharmaceutical projects are membrane proteins. Unfortunately, their structures are difficult to determine experimentally. We fill some of this structural void by developing computational methods of analyzing sequences and developing structural models of membrane proteins. We use computational analyses to do the following: 1) Address questions that are not answered by crystal structures. 2) Assist in understanding similarities and differences among homologous proteins. 3) Relate structural and sequence information to functional properties. 4) Assist in the design and interpretation of experimental studies. Our current projects can be classified into three areas: 1) models of the structure and gating mechanisms of potassium (K+) channels and their relatives; 2) models of the structure and gating mechanisms of the large mechanosensitive channel, MscL; and 3) development of methods to analyze sequences and construct structural models of membrane proteins. Project 1. Models of K+ Channels and Their Relatives K+ channels and other channels and transporters that evolved from K+ channels comprise one of the largest and most diverse groups of membrane proteins. These proteins are found in almost all cells from bacteria on up. This category of membrane proteins contains several diverse superfamilies of channels including Na+, Ca2+, cyclic nucleotide-gated, TRP and its homologs, glutamate-activated, and Ca2+ release channels plus some K+ symporters and transporters. The smallest of these proteins are 2TM K+ channels that have four identical subunits; each of which has only two transmembrane helices, M1 and M2. A 'P' hairpin segment that spans only the outer half of the transmembrane region is located between M1 and M2. The P segment determines the selectivity of the channel. 6TM K+ channels are more complex, with each alpha subunit having four additional transmembrane segments, S1-S4, that precede the pore-forming S5-P-S6 motif (analogous to the M1-P-M2 motif of 2TM channels) and that forms a voltage-sensing domain in voltage-gated channels. Voltage-gated Ca2+ and Na+ channels have only one alpha subunit; however, it contains four homologous 6TM motifs. The importance of understanding the structure and functional mechanisms of K+ channels was recognized this year by the awarding of the Nobel Prize in Chemistry to Roderick MacKinnon for his work in solving the crystal structures of two 2TM (KcsA and MthK) and one 6TM (KvAP) K+ channels. An additional 2TM channel structure, KirBac1.1, has also been determined recently. The effort to determine the KirBac1.1 structure was stimulation by our identification of this bacterial homolog of eukaryotic inward rectifying K+ channels. All of these structures are from prokaryotes. We are utilizing these crystallographic data in developing structural models of the gating mechanisms of both the crystallized prokaryotic proteins and of some of their eukaryotic homologs that have been studied extensively and that are important drug targets. The KvAP structure presents a particularly interesting molecular modeling challenge. It is difficult to reconcile the crystal structure of the complete KvAP channel protein and the paddle model of the voltage-dependent gating that MacKinnon's group developed based on this structure with many experimental results and with basic principles of membrane protein energetics. We suspect that the voltage-sensing domain (S1-S4) of this structure is grossly distorted, but that a second crystal structure of an isolated voltage-sensing domain has a native open conformation. We have developed alternative models of the open KvAP channel's structure by attaching the isolated voltage-sensing domain crystal structure to the pore domain (S5-P-S6) from the crystal structure of the complete protein. Using this model as a starting point, we have developed models of resting and intermediate conformations. Our models have the 'traditional' transmembrane topology in which each of the S1-S4 segments transverses the entire transmembrane region in all conformations. Much of the movement of S4 occurs via the helical screw mechanism. We were the first group to propose this topology and gating mechanism shortly after the first voltage-gated Na+ channel sequence was determined in the mid '80's. Molecular dynamics simulations that we have performed of the protein embedded in a lipid bilayer indicate that our models are substantially more stable than is the crystal structure of the complete KvAP protein. Our models of the KvAP channel were constrained to be consistent with experimental results from other Kv channels, primarily the Shaker channel. These constraints are complicated by the facts that these proteins are evolutionarily distant and substantial data have been obtained from Shaker residue positions that are deleted or that may be in a different conformation in the KvAP sequence. To better address these issues, we have developed models of the Shaker channel that are similar to those of our KvAP models, but with some important structural differences. These adjustments make the Shaker models consistent with many experimental observations that are inconsistent with MacKinnon's paddle model of gating. We are using the general structure of our models of the KvAP and Shaker channels in different conformations to model the general backbone folding and gating mechanism of another important class of K+ channels, the Herg channels. Our alignment of the Herg, KvAP, and Shaker sequences is based upon a very large multisequence alignment of all voltage-gated and cyclic nucleotide-gated 6TM channels and upon analyses of correlated mutations among different protein families. We use results of mutagenesis experiments on Herg and closely related EAG channels, results of NMR studies, plus basic modeling principles, to adjust features of these models and to model regions, such as the long S5-P loop in Herg channels for which analogous residues are deleted in KvAP. We are also modeling how the BeKm-1 scorpion toxin binds in the outer vestibule of Herg. The Herg project is being performed in collaboration with Dr. Gea-Ny Tseng, whose lab performs mutagenesis experiments that constrain and test our models. We have also been developing structural models of inward rectifying (Kir) potassium channels. Our project on Kir channels was nearing completion when the crystal structures of KirBac1.1 and KvAP were reported in April. While numerous features of our Kir models were supported by the KirBac1.1 structure, other features need modification. We decided to postpone our modeling efforts on Kir's until we completed our initial modeling of KvAP and the related Shaker and Herg channels. We are currently preparing manuscripts on the KvAP and Shaker models and will return to the Kir models soon. Project 2: Models of the Mechanosensitive Channel, MscL This project exemplifies our general approach to modeling the structures and functional mechanisms of membrane proteins. We have modeled the structure of the prokaryote mechanosensitive channel, MscL, as it undergoes a very large conformational change from a closed conformation to an open pore with a diameter greater than 30. This project began with the crystal structure of TbMscL, from M. tuberculosis, which was in a closed conformation.

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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Basic Sciences
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Tseng, Gea-Ny; Guy, H Robert (2005) Structure-function studies of the outer mouth and voltage sensor domain of hERG. Novartis Found Symp 266:19-35; discussion 35-45
Guy, H Robert (2005) Transmembrane interactions of alpha/beta integrin signaling. Structure 13:683-4
Durell, Stewart R; Shrivastava, Indira H; Guy, H Robert (2004) Models of the structure and voltage-gating mechanism of the shaker K+ channel. Biophys J 87:2116-30
Shafrir, Yinon; Guy, H Robert (2004) STAM: simple transmembrane alignment method. Bioinformatics 20:758-69
Shrivastava, Indira H; Durell, Stewart R; Guy, H Robert (2004) A model of voltage gating developed using the KvAP channel crystal structure. Biophys J 87:2255-70
Durell, S R; Bakker, E P; Guy, H R (2000) Does the KdpA subunit from the high affinity K(+)-translocating P-type KDP-ATPase have a structure similar to that of K(+) channels? Biophys J 78:188-99
Cho, H C; Tsushima, R G; Nguyen, T T et al. (2000) Two critical cysteine residues implicated in disulfide bond formation and proper folding of Kir2.1. Biochemistry 39:4649-57