Position Of Helix Research Articles

Design, synthesis and conformational analyses of bifacial benzamide based foldamers

Two bifacial oligobenzamide based scaffolds that mimic the side chains at i, i + 3 and i + 4 positions of an alpha helix are presented.

Dissecting the Inhibitory and Potentiation Effects of Desformylflustrabromine on a Muscle-Type Nicotinic Acetylcholine Receptor (NACHR)

Desformylflustrabromine (dFBr) potentiates 4β2 nAChR in vitro (Kim et al. 2007, Biorg. Med. Chem. Lett. 17: 4855) and reduces nicotine self-administration in vivo (Liu 2013; Psychopharmacology 230: 203). We have reported that dFBr is a potent inhibitor (IC50, ∼1 M) of human (Hα12β1eδ) and Torpedo (Tα12β1γδ) muscle-type nAChRs. We also found, using [3H]dFBr reversible binding and dFBr displacement of [3H]PCP binding, that dFBr binds with high affinity to the muscle-type nAChRs ion channel in the desensitized state. But, when we photolabeled nAChR-rich Torpedo membranes with [3H]dFBr in the presence of agonist, four dFBr binding sites were evident. [3H]dFBr photolabeled amino acids in the ion channel and in three binding sites within the extracellular domain previously identified for nAChRs PAMs galanthamine and physostigmine. The high affinity binding of dFBr in the ion channel precludes the ability to study the functional contribution of dFBr binding within the extracellular domain and to determine whether dFBr acts as inhibitor or potentiator at these sites. Therefore, we are introducing mutations in the muscle-type nAChR ion channel M2 helices to eliminate the channel blocking effect of dFBr. The effect of M2 mutations on the EC50 of ACh and on dFBr inhibition of muscle-type nAChR were examined using two-electrode voltage clamp recording. Serine substitutions of the leucine at M2-9 of each nAChR subunit decreased dFBr IC50 compared with wild-type, consistent with an open channel blocking mechanism. This results confirm our [3H]dFBr the photolabeling results demonstrating that dFBr binds within the ion channel in close proximity to M2-9. The effects of other substitutions at M2-9 and at other positions in the M2 helices on dFBr inhibition of muscle-type nAChR are being tested.

Computational de novo design of a four-helix bundle protein--DND_4HB.

The de novo design of proteins is a rigorous test of our understanding of the key determinants of protein structure. The helix bundle is an interesting de novo design model system due to the diverse topologies that can be generated from a few simple α-helices. Previously, noncomputational studies demonstrated that connecting amphipathic helices together with short loops can sometimes generate helix bundle proteins, regardless of the bundle's exact sequence. However, using such methods, the precise positions of helices and side chains cannot be predetermined. Since protein function depends on exact positioning of residues, we examined if sequence design tools in the program Rosetta could be used to design a four-helix bundle with a predetermined structure. Helix position was specified using a folding procedure that constrained the design model to a defined topology, and iterative rounds of rotamer-based sequence design and backbone refinement were used to identify a low energy sequence for characterization. The designed protein, DND_4HB, unfolds cooperatively (Tm >90°C) and a NMR solution structure shows that it adopts the target helical bundle topology. Helices 2, 3, and 4 agree very closely with the design model (backbone RMSD = 1.11 Å) and >90% of the core side chain χ1 and χ2 angles are correctly predicted. Helix 1 lies in the target groove against the other helices, but is displaced 3 Å along the bundle axis. This result highlights the potential of computational design to create bundles with atomic-level precision, but also points at remaining challenges for achieving specific positioning between amphipathic helices.

S-7 : A new cytokine receptor activation paradigm: Activation of JAK2 by the growth hormone receptor

The conformational changes required to transmit the GH binding signal from the extracellular domain of the GH receptor to its intracellular domain resulting in activation of JAK2 has been enigmatic. We have recently defined the first complete mechanistic model for JAK2 activation based on an archetypal cytokine receptor, the growth hormone receptor. To formulate this model we have used FRET to monitor positioning of the JAK2 binding motif, Jun-zipper receptor constructs to control receptor transmembrane (TM) helix position, atomistic modeling of TM helix interactions and docking of JAK2 kinase and inhibitory pseudokinase crystal structures with an opposing pair in trans [1] . Surprisingly, we found that activation of the receptor dimer induces a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel TM helix pair to a left handed crossover arrangement. This mechanism leads to removal of the pseudokinase domain from the kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans -activation. This model may generalize to other class I cytokine receptors.

Combination of genetic screening and molecular dynamics as a useful tool for identification of disease-related mutations: ZASP PDZ domain G54S mutation case.

Cypher/ZASP (LDB3 gene) is known to interact with a network of proteins. It binds to α-actinin and the calcium voltage channels (LTCC) via its PDZ domain. Here we report the identification of a highly conserved ZASP G54S mutation classified as a variant of unknown significance in a sample of an adult with hypertrophic cardiomyopathy (HCM). The initial bioinformatics calculations strongly evaluated G54S as damaging. Furthermore, we employed accelerated and classical molecular dynamics and free energy calculations to study the structural impact of this mutation on the ZASP apo form and to address the question of whether it can be linked to HCM. Seventeen independent MD runs and simulations of 2.5 μs total were performed and showed that G54S perturbs the α2 helix position via destabilization of the adjacent loop linked to the β5 sheet. This also leads to the formation of a strong H-bond between peptide target residues Leu17 and Gln66, thus restricting both the α-actinin2 and LTCC C-terminal peptides to access their natural binding site and reducing in this way their binding capacity. On the basis of these observations and the adult's clinical data, we propose that ZASP(G54S) and presumably other ZASP PDZ domain mutations can cause HCM. To the best of our knowledge, this is the first reported ZASP PDZ domain mutation that might be linked to HCM. The integrated workflow used in this study can be applied for the identification and description of other mutations that might be related to particular diseases.

Probing the Structure of the Mechanosensitive Channel of Small Conductance in Lipid Bilayers with Pulsed Electron-Electron Double Resonance

Mechanosensitive channel proteins are important safety valves against osmotic shock in bacteria, and are involved in sensing touch and sound waves in higher organisms. The mechanosensitive channel of small conductance (MscS) has been extensively studied. Pulsed electron-electron double resonance (PELDOR or DEER) of detergent-solubilized protein confirms that as seen in the crystal structure, the outer ring of transmembrane helices do not pack against the pore-forming helices, creating an apparent void. The relevance of this void to the functional form of MscS in the bilayer is the subject of debate. Here, we report PELDOR measurements of MscS reconstituted into two lipid bilayer systems: nanodiscs and bicelles. The distance measurements from multiple mutants derived from the PELDOR data are consistent with the detergent-solution arrangement of the protein. We conclude, therefore, that the relative positioning of the transmembrane helices is preserved in mimics of the cell bilayer, and that the apparent voids are not an artifact of detergent solution but a property of the protein that will have to be accounted for in any molecular mechanism of gating.

Glutathionylation of the Na K Pump

Reversible oxidative modifications of proteins are of importance for the normal cell functioning, e.g. for the receptor-coupled signaling. The formation of a disulfide bond between a protein and a glutathione tripeptide (Glu-Cys-Gly,) is an oxidative, posttranslational modification that might be involved in the cellular signaling. Recently, the Cys46 residue of the sarcolemmal Na K ATPAse β1 subunit has been proposed as a target for glutathionylation. Here, we use all-atom Molecular Dynamics (MD) simulations to investigate structural consequences of the Cys46 glutathionylation in the E2P state of the protein. Being negatively charged at physiological pH, the glutathione modification can induce alterations similar to the effect of phosphorylation. In contrast with previous studies, we find that Cys46, buried deeply in the membrane, can be exposed to the cytosolic glutathione due to the defect and local rearrangement of the protein-membrane interface in the E2P state, rather than the β1 helix sliding outside the membrane, previously anticipated for the E1 state. These findings are in accord with the recent crystal structure of the E1P state of Na+, K+-ATPase, where the position of the β1 helix is essentially the same as it is in the E2P state. We will comment on the accessibility of glutathione to Cys46, which lies at the center of the membrane.

The Effect of Interhelical Loop Length and Composition on the Electrostatic Interactions of the Voltage Sensor Domain of JShak1

Voltage gated potassium ion (Kv) channels are trans-membrane proteins that open and close their ion selective pore in response to changes in membrane voltage. Three negatively charged residues in the S2 and S3 helices are conserved in most Kv channels and form an electrostatic interaction network with positively charged residues of the S4, voltage sensing, helix. Previous studies with mouse Kv1.2 have shown that the length and composition of the S3-S4 linker are important in shaping the voltage response of Kv channels. To analyze how this S3-S4 loop effect has been adapted in a different lineage of the channel, we have studied the Kv1-family channel jShak1 from the jellyfish Polyorchis penicillatus. In jShak1 the conserved negatively charged glutamate residue in the S2 helix (position 227) is replaced by neutral asparagine. jShak1 has one less positively charged motif in the S4 helix and a very short S3-S4 linker, consisting of only five amino acid residues. Previous studies with jShak1 indicate that these factors might be responsible for constraining the interactions between the charged residues of the voltage sensing domain by limiting the movement of the S4 helix. We have replaced the natural short loop of jShak1 with different homo-polymers, varying both in length and composition and have mutated the last positively charged residue (K) in both natural and synthetic homo-loops. N227D and N227E mutations were combined with all synthetic loops to make double mutant channels. The homo-loops without a negatively charged residue at 227 do not shift the equilibrium significantly, but that the nature of the C-terminal residue alone can affect the V50 and both the V50 and the Boltzmann slope factor, when combined with a negatively charged residue at 227.

Mapping of the local environmental changes in proteins by cysteine scanning.

Protein conformational changes, which regulate the activity of proteins, are induced by the alternation of intramolecular interactions. Therefore, the detection of the local environmental changes around the key amino acid residues is essential to understand the activation mechanisms of functional proteins. Here we developed the methods to scan the local environmental changes using the vibrational band of cysteine S-H group. We validated the sensitivity of this method using bathorhodopsin, a photoproduct of rhodopsin trapped at liquid nitrogen temperature, which undergoes little conformational changes from the dark state as shown by the X-ray crystallography. The cysteine residues were individually introduced into 15 positions of Helix III, which contains several key amino acid residues for the light-induced conformational changes of rhodopsin. The shifts of S-H stretching modes of these cysteine residues and native cysteine residues upon the formation of bathorhodopsin were measured by Fourier transform infrared spectroscopy. While most of cysteine residues demonstrated no shift of S-H stretching mode, cysteine residues introduced at positions 117, 118, and 122, which are in the vicinity of the chromophore, demonstrated the significant changes. The current results are consistent with the crystal structure of bathorhodopsin, implying that the cysteine scanning is sensitive enough to detect the tiny conformational changes.

Investigation of Homeodomain Membrane Translocation Properties: Insights from the Structure Determination of Engrailed-2 Homeodomain in Aqueous and Membrane-Mimetic Environments

In addition to their well-known DNA-binding properties, homeodomains have the ability to efficiently translocate across biological membranes through still poorly-characterized mechanisms. To date, most biophysical studies addressing the mechanisms of internalization have focused on small synthetic peptides rather than full-length globular homeodomains. In this work, we characterized the conformational properties of chicken Engrailed 2 homeodomain (En2HD) in aqueous solution and in membrane mimetic environments using circular dichroism, Trp fluorescence, and NMR spectroscopy. En2HD adopts a well-defined three-helical bundle fold in aqueous solution. The Trp-48 residue, which is critical for internalization, is fully buried in the hydrophobic core. Circular dichroism and fluorescence reveal that a conformational transition occurs in anionic lipid vesicles and in micelles. En2HD loses its native three-dimensional structure in micellar environments but, remarkably, near-native helical secondary structures are maintained. Long-range interactions could be detected using site-directed spin labels, indicating that the three helices do not adopt extended orientations. Noncovalent paramagnetic probes yielded information about helix positioning and unveiled the burial of critical aromatic and basic residues within the micelles. Our results suggest that electrostatic interactions with membranes may be determinant in inducing a conformational change enabling Trp-48 to insert into membranes.

Novel Conserved Genotypes Correspond to Antibiotic Resistance Phenotypes of E. coli Clinical Isolates

Current efforts to understand antibiotic resistance on the whole genome scale tend to focus on known genes even as high throughput sequencing strategies uncover novel mechanisms. To identify genomic variations associated with antibiotic resistance, we employed a modified genome-wide association study; we sequenced genomic DNA from pools of E. coli clinical isolates with similar antibiotic resistance phenotypes using SOLiD technology to uncover single nucleotide polymorphisms (SNPs) unanimously conserved in each pool. The multidrug-resistant pools were genotypically similar to SMS-3-5, a previously sequenced multidrug-resistant isolate from a polluted environment. The similarity was evenly spread across the entire genome and not limited to plasmid or pathogenicity island loci. Among the pools of clinical isolates, genomic variation was concentrated adjacent to previously reported inversion and duplication differences between the SMS-3-5 isolate and the drug-susceptible laboratory strain, DH10B. SNPs that result in non-synonymous changes in gyrA (encoding the well-known S83L allele associated with fluoroquinolone resistance), mutM, ligB, and recG were unanimously conserved in every fluoroquinolone-resistant pool. Alleles of the latter three genes are tightly linked among most sequenced E. coli genomes, and had not been implicated in antibiotic resistance previously. The changes in these genes map to amino acid positions in alpha helices that are involved in DNA binding. Plasmid-encoded complementation of null strains with either allelic variant of mutM or ligB resulted in variable responses to ultraviolet light or hydrogen peroxide treatment as markers of induced DNA damage, indicating their importance in DNA metabolism and revealing a potential mechanism for fluoroquinolone resistance. Our approach uncovered evidence that additional DNA binding enzymes may contribute to fluoroquinolone resistance and further implicate environmental bacteria as a reservoir for antibiotic resistance.

Understanding the Role of Amphipathic Helices in N-BAR Domain Driven Membrane Remodeling

Endophilin N-BAR (N-terminal helix and Bin/amphiphysin/Rvs) domain tubulates and vesiculates lipid membranes in vitro via its crescent-shaped dimer and four amphipathic helices that penetrate into membranes as wedges. Like F-BAR domains, endophilin N-BAR also forms a scaffold on membrane tubes. Unlike F-BARs, endophilin N-BARs have N-terminal H0 amphipathic helices that are proposed to interact with other N-BARs in oligomer lattices. Recent cryo-electron microscopy reconstructions shed light on the organization of the N-BAR lattice coats on a nanometer scale. However, because of the resolution of the reconstructions, the precise positioning of the amphipathic helices is still ambiguous. In this work, we applied a coarse-grained model to study various membrane remodeling scenarios induced by endophilin N-BARs. We found that H0 helices of N-BARs prefer to align in an antiparallel manner at two ends of the protein to form a stable lattice. The deletion of H0 helices causes disruption of the lattice. In addition, we analyzed the persistence lengths of the protein-coated tubes and found that the stiffness of endophilin N-BAR-coated tubules qualitatively agrees with previous experimental work studying N-BAR-coated tubules. Large-scale simulations on membrane liposomes revealed a systematic relation between H0 helix density and local membrane curvature fluctuations. The data also suggest that the H0 helix is required for BARs to form organized structures on the liposome, further illustrating its important function.

Preparation of a new electrochemical biosensor for single base mismatch detection in DNA

In this study, a sandwich-type electrochemical DNA hybridization biosensor for the detection of single base mismatches (SBMs) has been described. 2,5-Dihydroxybenzoic acid (DHBA) is employed as a new redox probe in this study. For construction of the biosensor, thiolated capture DNA is self-assembled on a gold electrode surface and hybridized with one region of a target DNA sequence. Subsequently, a signaling amino-labeled probe DNA is hybridized to a second region of the target DNA to complete the DNA double-helix self-assembled monolayer (SAM). Finally DHBA is covalently attached to the signaling probe DNA using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) cross-linking reaction. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) are employed to characterize the modification steps. The electrochemical signal of DHBA was followed for studying different types of mismatches (GA, GT, TT, CA), both near the electrode surface (N-type) and far from it (F-type). The presence of a SBM leads to a considerable decrease in the electrochemical signal of DHBA compared to that of complementary DNA (cDNA), which makes the detection of mismatches (including thermodynamically stable GA mismatches) possible. The redox signal of DHBA was compared with that of methylene blue (MB) as a well-known and standard redox reporter in DNA biosensors. The results showed that for an N-type SBM, which is located between the electrode and the site of intercalation, the redox signal of MB switches off, whereas for an F-type SBM, which is located above the site of intercalation, electrons flow from the Au electrode to the intercalated MB, and hence the biosensor could not distinguish the presence and absence of an SBM. The proposed biosensor is straightforward with no need for target labeling, and is sensitive enough to detect SBMs independent of their position in DNA double helix.

Exploring the Mechanism of Cyclic Nucleotide Activation in the MLOTIK1 Potassium Channel

Cyclic nucleotides are essential elements in the cellular responses to hormones, light and smell. These molecules bind to their receptors through a well conserved cyclic nucleotide binding (CNB) domain, propagating a conformational change to an effector domain. We seek to understand the molecular details of the cyclic nucleotide activation mechanism. Our model protein is the CNB domain of a cyclic nucleotide regulated potassium channel from Mesorhizobium loti, a soil bacterium. This CNB domain is formed by a β-roll and three α-helices, named αA, αB and αC. A major difference between the bound and unbound states is the relative position of αC helix, which raises the hypothesis that αC helix motion could be the primary event in cyclic nucleotide binding.To address the hypothesis, we have monitored the conformational change in the isolated CNB domain. We tested two mutants having a single cysteine in αB or αC helix using a cysteine-reacting probe. Reaction kinetics were quantified by determining rate constants, which reflect the relative exposure of the cysteine. The cyclic nucleotides tested were not all equivalent in their rate constants. Moreover, the ratios between rate constants determined in the same conditions were different for αB and αC helix mutants. This indicates that ligand binding does not have the same effect on the two connected helices or, in other words, that the conformational change in αB helix is not totally dependent on that in αC helix. Therefore, the primary event in ligand binding is not the αC helix motion. Accordingly, point mutations in a functionally relevant residue in αC helix affected activity of the full-length channel only partially, suggesting that residues outside this helix must be involved in the activation mechanism.

Awakening Loss-of-Function KATP Channel Mutants with an Engineered ‘Forced Gating’ Approach

Regulation of inwardly-rectifying potassium channels by intracellular ligands couples membrane excitability to important signaling cascades and metabolic pathways. A significant barrier to understand coupling mechanisms between ligand binding and gating is that many functionally important channel motifs are highly sensitive to mutagenesis, resulting in a loss of function phenotype. We have developed a ‘forced gating’ approach that rescues function in electrically silent channel mutants, enabling characterization of channel motifs that are otherwise intractable to electrophysiological recording. This approach involves substitution of a glutamate in the hydrophobic Kir channel bundle crossing (F168E mutation in Kir6.2), generating channels that are pH sensitive and open upon alkalization, due to mutual repulsion of introduced negatively charged side chains in the channel gate. We have implemented this ‘forced gating’ approach in mutagenic scans of the Kir channel slide helix and G-loop, two motifs proposed to play a role in ligand dependent gating of Kir channels. Both motifs are also highly sensitive to mutagenesis, with alanine mutations causing nearly complete loss-of-function at 7/20 slide helix positions, and 8/13 G-loop positions. Without exception, expression of silent mutants on the Kir6.2[F168E] background permitted activation of functional channels in alkaline pH, and measurement of kinetics and potency of ATP inhibition. Our results highlight an essential ‘aspartate anchor’ (Kir6.2 residue D58) that bridges the slide helix and multiple interacting residues in the cytoplasmic domain. Disruption of the highly conserved ‘aspartate anchor’ uncouples the transmembrane and cytoplasmic domains, reducing ATP sensitivity of Kir6.2 far more than any other G-loop or slide helix mutants. These findings indicate a central role for the ‘aspartate anchor’ in coupling ligand binding to Kir gating, and also emphasize the potential general utility of this ‘forced gating’ method to study loss-of-function channel mutants.

Computational determination of the orientation of a heat repeat-like domain of DNA-PKcs

DNA dependent protein kinase catalytic subunit (DNA-PKcs) is an important regulatory protein in non-homologous end joining a process used to repair DNA double strand breaks. Medium resolution structures both from cryoEM and X-ray crystallography show the general topology of the protein and positions of helices in parts of DNA-PKcs. EM-Fold, an algorithm developed for building protein models into medium resolution density maps has been used to generate models for the heat repeat-like “Ring structure” of the molecule. We were able to computationally corroborate placement of the N-terminus of the domain that supports a previously published hypothesis. Targeted experiments are suggested to test the model.

Native State Conformational Heterogeneity of HP35 Revealed by Time-Resolved FRET

The villin headpiece subdomain (HP35) has become one of the most widely used model systems in protein folding studies, due to its small size and ultrafast folding kinetics. Here, we use HP35 as a test bed to show that the fluorescence decay kinetics of an unnatural amino acid, p-cyanophenylalanine (Phe(CN)), which are modulated by a nearby quencher (e.g., tryptophan or 7-azatryptophan) through the mechanism of fluorescence resonance energy transfer (FRET), can be used to detect protein conformational heterogeneity. This method is based on the notion that protein conformations having different donor-acceptor distances and interconverting slowly compared to the fluorescence lifetime of the donor (Phe(CN)) would exhibit different donor fluorescence lifetimes. Our results provide strong evidence suggesting that the native free energy basin of HP35 is populated with conformations that differ mostly in the position and mean helicity of the C-terminal helix. This finding is consistent with several previous experimental and computational studies. Moreover, this result holds strong implications for computational investigation of the folding mechanism of HP35.

Pore Determinants of KCNQ3 K+ Current Expression

KCNQ3 homomeric channels yield very small macroscopic currents compared with other KCNQ channels or KCNQ2/3 heteromers. Two disparate regions of the channels—the C-terminus and the pore region—have been implicated in governing KCNQ current amplitudes. We previously showed that the C-terminus plays a secondary role compared with the pore region. Here, we confirm the critical role of the pore region in determining KCNQ3 currents. We find that mutations at the 312 position in the pore helix of KCNQ3 (I312E, I312K, and I312R) dramatically decreased KCNQ3 homomeric currents as well as heteromeric KCNQ2/3 currents. Evidence that these mutants were expressed in the heteromers includes shifted TEA sensitivity compared with KCNQ2 homomers. To test for differential membrane protein expression, we performed total internal reflection fluorescence imaging, which revealed only small differences that do not underlie the differences in macroscopic currents. To determine whether this mechanism generalizes to other KCNQ channels, we tested the effects of analogous mutations at the conserved I273 position in KCNQ2, with similar results. Finally, we performed homology modeling of the pore region of wild-type and mutant KCNQ3 channels to investigate the putative structural mechanism mediating these results. The modeling suggests that the lack of current in I312E, I312K, and I312R KCNQ3 channels is due to pore helix-selectivity filter interactions that lock the selectivity filter in a nonconductive conformation.

Identification of a Crucial Amino Acid in the Helix Position 6.51 of Human Tachykinin Neurokinin 1 and 3 Receptors Contributing to the Insurmountable Mode of Antagonism by Dual NK1/NK3 Antagonists

The neurokinins are neuropeptides that elicit their effect through three GPCRs called NK(1), NK(2), and NK(3). Compounds 5 and 6 are dual hNK(1) (K(i) of 0.7 and 0.3 nM) and hNK(3) (K(i) of 2.9 and 1.7 nM) antagonists. Both compounds exhibit an insurmountable mode of antagonism at hNK(1), whereas at hNK(3), they differ in that 5 is an insurmountable but 6 a surmountable antagonist. Using homology modeling and site-directed mutagenesis, hNK(1)-Phe264 and hNK(3)-Tyr315 were found to be the molecular determinants of hNK(1) and hNK(3) antagonism by 5 and 6. In [(3)H]IP studies, the mutation hNK(1)-F264Y converted the mode of action of 5 from insurmountable to partial insurmountable antagonism while it had no effect on that of 6. Conversely, the mutation hNK(3)-Y315F enhanced the insurmountable behavior of 5 and converted 6's surmountable to an insurmountable antagonism. This finding was further confirmed by characterizing additional derivatives of 5 and 6, most notably with a hybrid structure.

Evolutionary analysis of the segment from helix 3 through helix 5 in vertebrate progesterone receptors

The interaction between helix 3 and helix 5 in the human mineralocorticoid receptor [MR], progesterone receptor [PR] and glucocorticoid receptor [GR] influences their response to steroids. For the human PR, mutations at Gly-722 on helix 3 and Met-759 on helix 5 alter responses to progesterone. We analyzed the evolution of these two sites and the rest of a 59 residue segment containing helices 3, 4 and 5 in vertebrate PRs and found that a glycine corresponding to Gly-722 on helix 3 in human PR first appears in platypus, a monotreme. In lamprey, skates, fish, amphibians and birds, cysteine is found at this position in helix 3. This suggests that the cysteine to glycine replacement in helix 3 in the PR was important in the evolution of mammals. Interestingly, our analysis of the rest of the 59 residue segment finds 100% sequence conservation in almost all mammal PRs, substantial conservation in reptile and amphibian PRs and divergence of land vertebrate PR sequences from the fish PR sequences. The differences between fish and land vertebrate PRs may be important in the evolution of different biological progestins in fish and mammalian PR, as well as differences in susceptibility to environmental chemicals that disrupt PR-mediated physiology.