Brian L. Lee

Structural and Functional Analysis of Transmembrane XI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na+/H+ exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H+ in exchange for one extracellular Na+. We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227–249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp238, Pro239, and Glu247. Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe230, Gly231, Ala236, Val237, Ala244, Val245, and Glu248 were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229–236 and 239–250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn227, Ile233, and Leu243 lining the translocation pore.

Structural and functional analysis of TM XI of the nhe1 isoform of the NA+/H+ exchanger

The Na+/H+ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals by extruding an intracellular H+ in exchange for one extracellular Na+. We characterized structural and functional aspects of the critical transmembrane (TM) segment XI (residues 449-470) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM XI was mutated to cysteine in the background of the cysteine-less protein and the sensitivity to water-soluble sulfhydryl reactive compounds MTSET ((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES ((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues with at least moderate activity remaining. Of the residues tested, only proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The activity of the L465C mutant was almost completely eliminated, whereas that of the L457C and I461C mutants was partially affected. The structure of a peptide representing TM XI (residues Lys447-Lys472) was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. The structure consisted of helical regions between Asp447-Tyr454 and Phe460-Lys471 at the N and C termini of the peptide, respectively, connected by a region with poorly defined, irregular structure consisting of residues Gly455-Gly459. TM XI of NHE1 had a structural similarity to TM XI of the Escherichia coli Na+/H+ exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with residue Leu465 contributing to the pore.

The Mechanism of Hsp90 ATPase Stimulation by Aha1

Hsp90 is a dimeric molecular chaperone responsible for the folding, maturation, and activation of hundreds of substrate proteins called ‘clients’. Numerous co-chaperone proteins regulate progression through the ATP-dependent client activation cycle. The most potent stimulator of the Hsp90 ATPase activity is the co-chaperone Aha1p. Only one molecule of Aha1p is required to fully stimulate the Hsp90 dimer despite the existence of two, presumably identical, binding sites for this regulator. Using ATPase assays with Hsp90 heterodimers, we find that Aha1p stimulates ATPase activity by a three-step mechanism via the catalytic loop in the middle domain of Hsp90. Binding of the Aha1p N domain to the Hsp90 middle domain exerts a small stimulatory effect but also drives a separate conformational rearrangement in the Hsp90 N domains. This second event drives a rearrangement in the N domain of the opposite subunit and is required for the stimulatory action of the Aha1p C domain. Furthermore, the second event can be blocked by a mutation in one subunit of the Hsp90 dimer but not the other. This work provides a foundation for understanding how post-translational modifications regulate co-chaperone engagement with the Hsp90 dimer.

Structural analysis of the Na+/H+ exchanger isoform 1 (NHE1) using the divide and conquer approach.

The sodium/proton exchanger isoform 1 (NHE1) is an ubiquitous plasma membrane protein that regulates intracellular pH by removing excess intracellular acid. NHE1 is important in heart disease and cancer, making it an attractive therapeutic target. Although much is known about the function of NHE1, current structural knowledge of NHE1 is limited to two conflicting topology models: a low-resolution molecular envelope from electron microscopy, and comparison with a crystal structure of a bacterial homologue, NhaA. Our laboratory has used high-resolution nuclear magnetic resonance (NMR) spectroscopy to investigate the structures of individual transmembrane helices of NHE1 - a divide and conquer approach to the study of this membrane protein. In this review, we discuss the structural and functional insights obtained from this approach in combination with functional data obtained from mutagenesis experiments on the protein. We also compare the known structure of NHE1 transmembrane segments with the structural and functional insights obtained from a bacterial sodium/proton exchanger homologue, NhaA. The structures of regions of the NHE1 protein that have been determined have both similarities and specific differences to the crystal structure of the NhaA protein. These have allowed insights into both the topology and the function of the NHE1 protein.

Structural and functional analysis of extracellular loop 2 of the Na(+)/H(+) exchanger.

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pHi) by removing one intracellular H(+) in exchange for one extracellular Na(+). It has a large 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments and several membrane-associated segments. Extracellular regions of this domain are believed to contribute to cation coordination, transport and sensitivity to inhibitors. In this study we characterized the structure and function of extracellular loop 2. Mutation of residues Pro153, Pro154 and Phe155 demonstrated that these residues were critical for efficient NHE1 function. Mutations to Ala resulted in decreases in cation affinity and in decreases in activity of the protein, these were more marked in both Pro154 and Phe155. NMR spectroscopy was used to characterize the solution structure of a peptide NAc-Gly150-Phe155-NH(2). The peptide showed at least three different conformers in solution due to cis-trans isomerization of the Thr(152)-Pro(153) and Pro(153)-Pro(154) peptide bonds. The trans-trans conformation appeared to be in an extended conformation, whereas the cis-trans conformation showed a propensity to form a beta turn. Our results show that the EL2 region is critical to NHE1 function and that a peptide of the EL2 region can adopt different structures in solution potentially forming a beta turn that is important in function of the full protein Mutation of Pro(154) could disrupt the beta turn, affecting helix packing and the protein structure and function.

Structural and functional analysis of critical amino acids in TMVI of the NHE1 isoform of the Na+/H+ exchanger

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) resides on the plasma membrane and exchanges one intracellular H(+) for one extracellular Na(+). It maintains intracellular pH and regulates cell volume, and cell functions including growth and cell differentiation. Previous structural and functional studies on TMVI revealed several amino acids that are potentially pore lining. We examined these and other critical residues by site-directed mutagenesis substituting Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp, Gln; Glu248→Asp, Gln. Mutant NHE1 proteins were characterized in AP-1 cells, which do not express endogenous NHE1. All the TMVI critical amino acids were highly sensitive to substitution and changes often lead to a dysfunctional protein. Mutations of Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp; Glu248→Gln yielded significant reduction in NHE1 activity. Mutants of Asn227 demonstrated defects in protein expression, targeting and activity. Substituting Asn227→Arg and Ile233→Ala decreased the surface localization and expression of NHE1 respectively. The pore lining amino acids Ile233 and Leu243 were both essential for activity. Glu247 was not essential, but the size of the residue at this location was important while the charge on residue Glu248 was more critical to NHE1 function. Limited trypsin digestion on Leu243→Ala and Glu248→Gln revealed that they had increased susceptibility to proteolytic attack, indicating an alteration in protein conformation. Modeling of TMVI with TMXI suggests that these TM segments form part of the critical fold of NHE1 with Ile233 and Leu465 of TMXI forming a critical part of the extracellular facing ion conductance pathway.

Structural and Functional Analysis of Transmembrane Segment IV of the Salt Tolerance Protein Sod2

Background: Sod2 is the salt tolerance protein of Schizosaccharomyces pombe. Results: Alanine scanning mutagenesis and structural analysis demonstrated that amino acids 144–147 are in an extended region of transmembrane segment IV and are critical for function. Conclusion: Mutations in transmembrane segment IV affect Sod2 cation transport. Significance: TM IV of Sod2 has conserved structures with other Na+/H+ exchangers important in salt tolerance. Sod2 is the plasma membrane Na+/H+ exchanger of the fission yeast Schizosaccharomyces pombe. It provides salt tolerance by removing excess intracellular sodium (or lithium) in exchange for protons. We examined the role of amino acid residues of transmembrane segment IV (TM IV) (126FPQINFLGSLLIAGCITSTDPVLSALI152) in activity by using alanine scanning mutagenesis and examining salt tolerance in sod2-deficient S. pombe. Two amino acids were critical for function. Mutations T144A and V147A resulted in defective proteins that did not confer salt tolerance when reintroduced into S. pombe. Sod2 protein with other alanine mutations in TM IV had little or no effect. T144D and T144K mutant proteins were inactive; however, a T144S protein was functional and provided lithium, but not sodium, tolerance and transport. Analysis of sensitivity to trypsin indicated that the mutations caused a conformational change in the Sod2 protein. We expressed and purified TM IV (amino acids 125–154). NMR analysis yielded a model with two helical regions (amino acids 128–142 and 147–154) separated by an unwound region (amino acids 143–146). Molecular modeling of the entire Sod2 protein suggested that TM IV has a structure similar to that deduced by NMR analysis and an overall structure similar to that of Escherichia coli NhaA. TM IV of Sod2 has similarities to TM V of the Zygosaccharomyces rouxii Na+/H+ exchanger and TM VI of isoform 1 of mammalian Na+/H+ exchanger. TM IV of Sod2 is critical to transport and may be involved in cation binding or conformational changes of the protein.

Structural and Functional Analysis of the Transmembrane Segment Pair VI and VII of the NHE1 Isoform of the Na(+)/H(+) Exchanger.

Isoform 1 of the mammalian Na(+)/H(+) exchanger (NHE1) is a ubiquitously expressed plasma membrane pH regulatory protein. It removes one intracellular H(+) in exchange for one extracellular Na(+). The 500 N-terminal amino acids comprise the catalytic membrane domain and fold into 12 transmembrane (TM) segments. To gain insight into the structure and function of human NHE1, a region spanning transmembrane domains VI and VII was expressed and purified, and the structure was determined using nuclear magnetic resonance (NMR). Segment VI includes two structurally conserved regions corresponding to two short α-helices involving residues 229-236 and 239-247. Segment VII includes one long helical region spanning residues 255-274. The NMR structure of the peptide containing transmembrane domains VI and VII was very similar to the previously published structures of the single-transmembrane segments except that TM VII was not kinked. Tryptophan scanning site-directed mutagenesis of TM VI demonstrated that mutation of residues V240-V245 to tryptophan eliminated NHE1 activity when the full length protein was expressed in cells. In contrast, mutants F246W and E247W were functional. Double mutant V242F/F260V retained activity, while the individual mutations were not active. The results suggest that the region of TM VI from V240 to V245 is closely associated with TM VII and that, in agreement with the NMR structure of VI-VII segments, V242 and F260 are in close association. A study of two transmembrane peptides provides further insight into the structure of the NHE1 protein.

Structural and functional analysis of extracellular loop 4 of the Nhe1 isoform of the Na + /H + exchanger

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein. It regulates intracellular pH by removing a single intracellular H(+) in exchange for one extracellular Na(+). The membrane domain of NHE1 comprises the 500 N-terminal amino acids and is made of 12 transmembrane segments. The extracellular loops of the transmembrane segments are thought to be involved in cation coordination and inhibitor sensitivity. We have characterized the structure and function of amino acids 278-291 representing extracellular loop 4. When mutated to Cys, residues F277, F280, N282 and E284 of EL4 were sensitive to mutation and reaction with MTSET inhibiting NHE1 activity. In addition they were found to be accessible to extracellular applied MTSET. A peptide of the amino acids of EL4 was mostly unstructured suggesting that it does not provide a rigid structured link between TM VII and TM VIII. Our results suggest that EL4 makes an extension upward from TM VII to make up part of the mouth of the NHE1 protein and is involved in cation selectivity or coordination. EL4 provides a flexible link to TM VIII which may either allow movement of TM VII or allow TM VIII to not be adjacent to TM VII.

Molecular Basis for K63-Linked Ubiquitination Processes in Double-Strand DNA Break Repair: A Focus on Kinetics and Dynamics

Cells are exposed to thousands of DNA damage events on a daily basis. This damage must be repaired to preserve genetic information and prevent development of disease. The most deleterious damage is a double-strand break (DSB), which is detected and repaired by mechanisms known as non-homologous end-joining (NHEJ) and homologous recombination (HR), which are components of the DNA damage response system. NHEJ is an error-prone first line of defense, whereas HR invokes error-free repair and is the focus of this review. The functions of the protein components of HR-driven DNA repair are regulated by the coordinated action of post-translational modifications including lysine acetylation, phosphorylation, ubiquitination, and SUMOylation. The latter two mechanisms are fundamental for recognition of DSBs and reorganizing chromatin to facilitate repair. We focus on the structures and molecular mechanisms for the protein components underlying synthesis, recognition, and cleavage of K63-linked ubiquitin chains, which are abundant at damage sites and obligatory for DSB repair. The forward flux of the K63-linked ubiquitination cascade is driven by the combined activity of E1 enzyme, the heterodimeric E2 Mms2-Ubc13, and its cognate E3 ligases RNF8 and RNF168, which is balanced through the binding and cleavage of chains by the deubiquitinase BRCC36, and the proteasome, and through the binding of chains by recognition modules on repair proteins such as RAP80. We highlight a number of aspects regarding our current understanding for the role of kinetics and dynamics in determining the function of the enzymes and chain recognition modules that drive K63 ubiquitination.