Peptide-like Molecules Research Articles

Annotation of DOM metabolomes with an ultrahigh resolution mass spectrometry molecular formula library

Increased accessibility of liquid chromatography mass spectrometry (LC-MS) metabolomics instrumentation and software have expanded their use in studies of dissolved organic matter (DOM) and exometabolites released by microbes. Current strategies to annotate metabolomes generally rely on matching tandem MS/MS spectra to databases of authentic standards. However, spectral matching approaches typically have low annotation rates for DOM. An alternative approach is to annotate molecular formula based on accurate mass and isotopic fine structure measurements that can be obtained from state-of-the-art ultrahigh resolution Fourier Transform Ion Cyclotron Resonance mass spectrometry (FT-ICR MS), but instrument accessibility for large metabolomic studies is generally limited. Here, we describe a strategy to annotate exometabolomes obtained from lower resolution LC-MS systems by matching metabolomic features to a molecular formula library generated for a representative sample analyzed by LC 21T- FT-ICR MS. The molecular formula library approach successfully annotated 53% of exometabolome features of the marine diatom Phaeodactylum tricornutum – a nearly ten-fold increase over the 6% annotation rate achieved using a conventional MS/MS approach. There was 94% agreement between assigned formula that were annotated with both approaches, and mass error analysis of the discrepancies suggested that the FT-ICR MS formula assignments were more reliable. Differences in the exometabolome of P. tricornutum grown under iron replete and iron limited conditions revealed 668 significant metabolites, including a suite of peptide-like molecules released by P. tricornutum in response to iron deficiency. These findings demonstrate the utility of FT-ICR MS formula libraries for extending the accuracy and comprehensiveness of metabolome annotations.

Residue profiles of peptides with cholesterol esterase and pancreatic lipase inhibitory activities through virtual screening and sequence analysis

The detailed characterization of the structural features of peptides targeting cholesterol esterase (CEase) or pancreatic lipase (PPL) will benefit the management of hyperlipidemia and obesity. This study employed the Glide SP (standard precision)-peptide method to predict the binding modes of 202 dipeptides and 203 tripeptides to these targets, correlating residue composition and position with binding energy. Strong preferences for Trp, Phe, and Tyr were observed at all positions of potential inhibitory peptides, whereas negatively charged residues Glu and Asp were disfavored. Notably, Arg and aromatic rings significantly influenced the peptide conformation at the active site. Tripeptide IWR demonstrated the high efficacy, with IC50 values of 0.214 mg/mL for CEase and 0.230 mg/mL for PPL. Five novel IWR scaffold-tetrapeptides exhibited promising inhibitory activity. Non-covalent interactions and energy contributions dominated the formation of stable complexes. Our results provide insights for the development of new sequences or peptide-like molecules with enhanced inhibitory activity.

Molecular-level insights into the temperature-dependent formation dynamics and mechanism of water-soluble dissolved organic carbon derived from biomass pyrolysis smoke

Water soluble organic carbon (WSOC) derived from biomass pyrolytic smoke is deposited through atmospheric aerosols, negatively affecting aquatic ecological quality and safety. However, the temperature-dependent molecular diversity and dynamic formation of smoke-derived WSOC remain poorly understood in water. Herein, we explored the molecular-level formation mechanism of pyrolytic smoke-derived WSOC in water to explain the evolution, heterogeneous correlations, and sequential responses of molecules and functional groups to increasing pyrolysis temperature. Two-dimensional correlation spectroscopy was used to innovatively establish the characteristic correlations between spectroscopy and Fourier transform-ion cyclotron resonance mass spectrometry. Temperature-dependent formation of WSOC exhibited diversity in absorbance/fluorescent components, unique/common molecules, and their chemical parameters, showing the simultaneous formation and degradation reactions. The common WSOC molecules with lower and higher degrees of oxidation showed significant positive and negative correlations with the fluorescent components, respectively. The primary sequential response of WSOC molecules to increasing pyrolysis temperature (lignin-like molecules → unsaturated hydrocarbons, condensed aromatic molecules → lipid-like/aliphatic-/peptide-like molecules) corresponded to the temperature response of functional groups (carboxylic/alcoholic → polysaccharides → aromatics/amides/phenolic/aliphatic groups), demonstrating well synergistic relationships between them. These novel findings will contribute to the comprehensive understanding and assessments of potential environmental behavior or risks of WSOC in aquatic ecosystems.

Mapping Observations of Peptide-like Molecules around Sagittarius B2

Peptide-like molecules, which have a close connection with the origin of life, have been detected in the Universe. Mapping observations of HCONH2 and CH3CONH2, two of the simplest peptide-like molecules, are performed toward the Sagittarius B2 (Sgr B2) complex with the IRAM 30 m telescope. Seven transitions of HCONH2 and five transitions of CH3CONH2 are used in the analysis. The spatial distributions of the excitation temperature and column density of HCONH2 in the molecular envelope of Sgr B2 are obtained by rotation diagrams. Assuming the same excitation temperature of HCONH2, the column densities of CH3CONH2 are also calculated. The results show that the excitation temperature ranges from 6 to 46 K in the molecular envelope of Sgr B2. The abundance ratios between HCONH2 and CH3CONH2 are calculated to explore the relationship between them, as are those between HCONH2 and HNCO. The abundance ratio of CH3CONH2/HCONH2 varies from 10% to 20%, while that of HCONH2/HNCO ranges from 1.5% to 10%. CH3CONH2 is enhanced with respect to HCONH2 in the northwest region of Sgr B2. One transition of H13CONH2 is detected toward 12 positions of Sgr B2, from which a 12C/13C ratio of 28.7 is obtained. A time-dependent chemical model with a short-duration X-ray burst is used to explain the observed abundances of HCONH2 and CH3CONH2, with the best-fitting result at T dust = 53–56 K. More chemical reactions are required to be included in the model since the modeled abundance is lower than the observed one at the observed T dust.

Tentative detection of cyanoformamide NCCONH2 in space

Abstract The peptide-like molecule cyanoformamide (NCCONH2) is the cyano (CN) derivative of formamide (NH2CHO). It is known to play a role in the synthesis of nucleic acid precursors under prebiotic conditions. In this paper, we present a tentative detection of NCCONH2 in the interstellar medium with the Atacama Large Millimeter/submillimeter Array (ALMA) archive data. 10 unblended lines of NCCONH2 were seen around 3σ noise levels toward Sagittarius B2(N1E), a position that is slightly offset from the continuum peak. The column density of NCCONH2 was estimated to be 2.4 × 1015 cm−2, and the fractional abundance of NCCONH2 toward Sgr B2(N1E) was 6.9 × 10−10. The abundance ratio between NCCONH2 and NH2CHO is estimated to be ∼0.01. We also searched for other peptide-like molecules toward Sgr B2(N1E). The abundances of NH2CHO, CH3NCO and CH3NHCHO toward Sgr B2(N1E) were about 1/10 of those toward Sgr B2(N1S), while the abundance of CH3CONH2 was only 1/20 of that toward Sgr B2(N1S).

Molecular transformation of organic nitrogen in Antarctic penguin guano-affected soil

Organic nitrogen (ON) is an important participant in the Earth’s N cycle. Previous studies have shown that penguin feces add an abundance of nutrients including N to the soil, significantly changing the eco-environment in ice-free areas in Antarctica. To explore the molecular transformation of ON in penguin guano-affected soil, we collected guano-free weathered soil, modern guano-affected soil from penguin colonies, ancient guano-affected soil from abandoned penguin colonies, and penguin feces from the Ross Sea region, Antarctica, and Fourier transform ion cyclotron mass spectrometry (FT-ICR MS) was used to investigate the chemical composition of water-extractable ON. By comparing the molecular compositions of ON among different samples, we found that the number of ON compounds (>4,000) in weathered soil is minimal, while carboxylic-rich alicyclic-like molecules (CRAM-like) are dominant. Penguin feces adds ON into the soil with > 10,000 CHON, CHONS and CHN compounds, including CRAM-like, lipid-like, aliphatic/ peptide-like molecules and amines in the guano-affected soil. After the input of penguin feces, macromolecules continue to degrade, and other ON compounds tend to be oxidized into relatively stable CRAM-like molecules, this is an important transformation process of ON in guano-affected soils. We conclude the roles of various forms of ON in the N cycle are complex and diverse. Combined with previous studies, ON eventually turns into inorganic N and is lost from the soil. The lost N ultimately returns to the ocean and the food web, thus completing the N cycle. Our study preliminarily reveals the molecular transformation of ON in penguin guano-affected soil and is important for understanding the N cycle in Antarctica.

Coarse-grained intrinsically disordered protein simulations with explicit ions parameterized by salting-out constants.

Implicit solvent coarse-grained models have emerged as an effective method for simulating intrinsically disordered proteins (IDPs) due to the long length and time-scale need to effectively simulate their equilibrium behavior. These coarse-grained methods typically rely on Debye-Hückel theory to explain the effects of salt in the simulations. However, Debye-Hückel electrostatics are only accurate over a limited range of ionic strengths and are also unable to capture ion-type specific effects, typically called Hofmeister effects that arise at higher salt concentrations. To address this, we developed an explicit ion model of potassium chloride that can interface with the otherwise implicit solvent hydrophobicity scale model for intrinsically disordered protein. Parameters for the explicit ion model were determined by matching the salting-out constants of several small peptide-like molecules. Salting-out constants were determined in the simulation by creating a system with an ion concentration gradient via an external potential and performing umbrella sampling along the concentration gradient. The remaining amino acids for which there is no data for the salting-out constant are determined using a scaled Kapcha-Rosky hydrophobicity scale determined from the umbrella sampling simulations. The parameters are validated against a set FRET data from a diverse set of linker peptides at several salt concentrations. This explicit ion model is able to capture the changes in protein expansion as a function of salt concentration at concentration above 0.5 M where Debye-Hückel screened electrostatics predict no change in protein expansion.

Mechanistic Insights into Angiotensin I-Converting Enzyme Inhibitory Tripeptides to Decipher the Chemical Basis of Their Activity.

Food proteins are an important source of bioactive peptides, and the angiotensin I-converting enzyme (ACE) inhibitors are worthy of attention for their possible beneficial effects in subjects with mild hypertension. However, the chemical basis underpinning their activity is not well-understood, hampering the discovery of novel inhibitory sequences from the plethora of peptides encrypted in food proteins. This work combined computational and in vitro investigations to describe precisely the chemical basis of potent inhibitory tripeptides. A substantial set of previously uncharacterized tripeptides have been investigated in silico and in vitro, and LCP was described for the first time as a potent ACE inhibitory peptide with IC50 values of 8.25 and 6.95 μM in cell-free and cell-based assays, respectively. The outcomes presented could serve to better understand the chemical basis of already characterized potent inhibitory tripeptides or as a blueprint to design novel and potent inhibitory peptides and peptide-like molecules.

Propionamide (C2H5CONH2): The Largest Peptide-like Molecule in Space

Abstract Peptide bonds, as the molecular bridges that connect amino acids, are crucial to the formation of proteins. Searches and studies of molecules with embedded peptide-like bonds are thus important for the understanding of protein formation in space. Here we report the first tentative detection of propionamide (C2H5CONH2), the largest peptide-like molecule detected in space toward Sagittarius B2(N1) at a position called N1E that is slightly offset from the continuum peak. New laboratory measurements of the propionamide spectrum were carried out in the 9–461 GHz range, which provide good opportunity to check directly for the transition frequencies of detected interstellar lines of propionamide. Our observing result indicates that propionamide emission comes from the warm, compact cores in Sagittarius B2, in which massive protostellars are forming. The column density of propionamide toward Sgr B2(N1E) was derived to be 1.5 × 1016 cm−2, which is three-fifths of that of acetamide, and one-nineteenth of that of formamide. This detection suggests that large peptide-like molecules can form and survive during star-forming process and may form more complex molecules in the interstellar medium (ISM). The detection of propionamide bodes well for the presence of polypeptides, as well as other complex prebiotic molecules in the ISM.

The GUAPOS project

Context. Peptide-like bond molecules, which can take part in the formation of proteins in a primitive Earth environment, have been detected only towards a few hot cores and hot corinos up to now. Aims. We present a study of HNCO, HC(O)NH2, CH3NCO, CH3C(O)NH2, CH3NHCHO, CH3CH2NCO, NH2C(O)NH2, NH2C(O)CN, and HOCH2C(O)NH2 towards the hot core G31.41+0.31. The aim of this work is to study these species together to allow a consistent study among them. Methods. We have used the spectrum obtained from the ALMA 3 mm spectral survey GUAPOS, with a spectral resolution of ~0.488 MHz (~1.3–1.7 km s−1) and an angular resolution of 1.′′2 × 1.′′2 (~4500 au), to derive column densities of all the molecular species presented in this work, together with 0.′′2 × 0.′′2 (~750 au) ALMA observations from another project to study the morphology of HNCO, HC(O)NH2, and CH3C(O)NH2. Results. We have detected HNCO, HC(O)NH2, CH3NCO, CH3C(O)NH2, and CH3NHCHO, but no CH3CH2NCO, NH2C(O)NH2, NH2C(O)CN, or HOCH2C(O)NH2. This is the first time that these molecules have been detected all together outside the Galactic centre. We have obtained molecular fractional abundances with respect to H2 from 10−7 down to a few 10−9 and abundances with respect to CH3OH from 10−3 to ~4 × 10−2, and their emission is found to be compact (~2′′, i.e. ~7500 au). From the comparison with other sources, we find that regions in an earlier stage of evolution, such as pre-stellar cores, show abundances at least two orders of magnitude lower than those in hot cores, hot corinos, or shocked regions. Moreover, molecular abundance ratios towards different sources are found to be consistent between them within ~1 order of magnitude, regardless of the physical properties (e.g. different masses and luminosities), or the source position throughout the Galaxy. Correlations have also been found between HNCO and HC(O)NH2 as well as CH3NCO and HNCO abundances, and for the first time between CH3NCO and HC(O)NH2, CH3C(O)NH2 and HNCO, and CH3C(O)NH2 and HC(O)NH2 abundances. These results suggest that all these species are formed on grain surfaces in early evolutionary stages of molecular clouds, and that they are subsequently released back to the gas phase through thermal desorption or shock-triggered desorption.

Synthesis of a novel category of pseudo-peptides using an Ugi three-component reaction of levulinic acid as bifunctional substrate, amines, and amino acid-based isocyanides.

The synthesis of a novel category of pseudo-peptides via intramolecular Ugi reaction of levulinic acid (4-oxopentanoic acid), aromatic and aliphatic amines, and amino acid-based isocyanides is reported. Levulinic acid was applied as a bifunctional substrate containing both carbonyl and acid moieties suitable for the Ugi reaction. This article provides a facile and convenient one-pot procedure for the synthesis of peptide-like heterocyclic molecules containing 2-pyrrolidone (γ-lactam), amide and ester functional groups with good to excellent yields.

Chemical modelling of complex organic molecules with peptide-like bonds in star-forming regions

Peptide bonds (N-C=O) play a key role in metabolic processes since they link amino acids into peptide chains or proteins. Recently, several molecules containing peptide-like bonds have been detected across multiple environments in the interstellar medium (ISM), growing the need to fully understand their chemistry and their role in forming larger pre-biotic molecules. We present a comprehensive study of the chemistry of three molecules containing peptide-like bonds: HNCO, NH$_2$CHO, and CH$_3$NCO. We also included other CHNO isomers (HCNO, HOCN), and C$_2$H$_3$NO isomers (CH$_3$OCN, CH$_3$CNO) to the study. We have used the \uclchem gas-grain chemical code and included in our chemical network all possible formation/destruction pathways of these peptide-like molecules recently investigated either by theoretical calculations or in laboratory experiments. Our predictions are compared to observations obtained toward the proto-star IRAS16293$-$2422 and the L1544 pre-stellar core. Our results show that some key reactions involving the CHNO and C$_2$H$_3$NO isomers need to be modified to match the observations. Consistently with recent laboratory findings, hydrogenation is unlikely to produce NH$_2$CHO on grain surfaces, while a combination of radical-radical surface reactions and gas-phase reactions is a better alternative. In addition, better results are obtained for NH$_2$CHO when a slightly higher activation energy of 25$\,$K is considered for the gas-phase reaction $\rm NH_2 + H_2CO \rightarrow NH_2CHO + H$. Finally, our modelling shows that the observed correlation between NH$_2$CHO and HNCO in star-forming regions may come from the fact that HNCO and NH$_2$CHO react to temperature in the same manner rather than from a direct chemical link between the two species.

Novel mixed vesicles containing lactobacilli biosurfactant for vaginal delivery of an anti-Candida agent

The purpose of this work was to prepare and characterize an innovative formulation for vaginal delivery of econazole nitrate, commonly used for the treatment of Candida infections. A novel biosurfactant isolated from a vaginal Lactobacillus strain was used to prepare phosphatidylcholine based mixed vesicles. Biosurfactant was produced by Lactobacillus gasseri BC9, isolated from the vagina of a healthy premenopausal woman, and was chemically characterized by FT-IR and ESI-MS. Mixed vesicles, obtained through film rehydration and extrusion method, were characterized in terms of size, zeta potential, encapsulation efficiency, mucoadhesion properties and econazole release. Moreover, the antimicrobial activity of the mixed vesicles was tested towards both planktonic cultures and biofilms of Candida albicans. Biosurfactant produced by L. gasseri BC9 was composed by peptide-like molecules containing hydrocarbon chains and possessed a high surface activity together with a low critical micelle concentration. All the mixed vesicles presented optimal diameter range (226–337nm) for topical vaginal administration. Econazole-loaded mixed vesicles containing biosurfactant showed higher encapsulation efficiency and mucoadhesion ability with respect to vesicles containing Tween 80. Further, they allowed a sustained release of econazole nitrate, maintaining the antifungal activity against C. albicans planktonic culture. Notably, biosurfactant-based vesicles were significantly more active than free econazole in the eradication of Candida biofilm. In conclusion, mixed vesicles are promising new vaginal delivery systems for the potential employment in the treatment of chronic infections.

Diversity-oriented synthesis of cyclopropyl peptides from Ugi-derived dehydroalanines

A three-step synthesis of cyclopropyl peptides is reported. The protocol involves a consecutive Ugi-4CR/elimination reaction to prepare dehydroalanines followed by a Corey-Chaykovsky cyclopropanation reaction. Peptide-like molecules that resemble some pharmacologically active compounds with a variety of substituents in the cyclopropane ring were prepared. When (2-ethoxy-2-oxoethyl) dimethyl sulfonium ylide was used the reaction exclusively gives the cis-diastereoisomer cyclopropanes in good yields from readily prepared starting materials. A collection of 26 highly substituted cyclopropyl peptides were obtained.

CH3NCO detections in observations and the laboratory

AbstractMethyl isocyanate (CH3NCO) belongs to a select group of peptide-like prebiotic molecules. In this paper we present its first detection toward the solar type low-mass protostar IRAS16293-2422 (hereafter IRAS16293). CH3NCO is detected towards IRAS16293 as a warm component with Tex > 100 K and HNCO/CH3NCO ∼4-12. Also, its grain surface formation route is investigated in the laboratory. VUV processing of CH4:HNCO mixtures, investigated by IR spectroscopy and mass spectrometry, revealed that it can be formed by reactions of CH3 and with (H)NCO. Observations and experiments strongly hint that methyl isocyanate is formed on interstellar dust grains.

Di- and tripeptide transport in vertebrates: the contribution of teleost fish models.

Solute Carrier 15 (SLC15) family, alias H+-coupled oligopeptide cotransporter family, is a group of membrane transporters known for their role in the cellular uptake of di- and tripeptides (di/tripeptides) and peptide-like molecules. Of its members, SLC15A1 (PEPT1) chiefly mediates intestinal absorption of luminal di/tripeptides from dietary protein digestion, while SLC15A2 (PEPT2) mainly allows renal tubular reabsorption of di/tripeptides from ultrafiltration, SLC15A3 (PHT2) and SLC15A4 (PHT1) possibly interact with di/tripeptides and histidine in certain immune cells, and SLC15A5 has unknown function. Our understanding of this family in vertebrates has steadily increased, also due to the surge of genomic-to-functional information from 'non-conventional' animal models, livestock, poultry, and aquaculture fish species. Here, we review the literature on the SLC15 transporters in teleost fish with emphasis on SLC15A1 (PEPT1), one of the solute carriers better studied amongst teleost fish because of its relevance in animal nutrition. We report on the operativity of the transporter, the molecular diversity, and multiplicity of structural-functional solutions of the teleost fish orthologs with respect to higher vertebrates, its relevance at the intersection of the alimentary and osmoregulative functions of the gut, its response under various physiological states and dietary solicitations, and its possible involvement in examples of total body plasticity, such as growth and compensatory growth. By a comparative approach, we also review the few studies in teleost fish on SLC15A2 (PEPT2), SLC15A4 (PHT1), and SLC15A3 (PHT2). By representing the contribution of teleost fish to the knowledge of the physiology of di/tripeptide transport and transporters, we aim to fill the gap between higher and lower vertebrates.

Electron-Induced Fragmentation of Methylated Formamides

At an electron ionization of 70eV, all cationic fragments for N,N-dimethylformamide (DMF) and N-methylformamide (NMF) were detected with a quadrupole mass spectrometer and confirmed using isotopically-labelled molecules. Ion efficiency curves near the threshold region (5–20eV) for all of the major cationic fragments were measured and fit with a Wannier-type function to determine their appearance energies (AEs). Possible reaction pathways for the formation of each observed cation were proposed, and the corresponding enthalpies (ΔH⁰s) of reaction were evaluated from quantum chemical calculations using the Gaussian09 computational quantum chemistry package. Our results suggest that hydrogen loss for both molecules occurs primarily from the carbonyl carbon. We confirm that hydrogen transfer from the carbonyl site to the nitrogen site occurs during amide bond cleavage. The resultant cations, C2H6N+ and CH4N+, and their further fragments, formed by losing an H atom, H2, or C2H2, were first identified in mass spectra and were further probed by comparing the AEs with calculated ΔH⁰s of all possible formation pathways. Additionally, we suggest that hydrogen transfer also occurs when the N-CH3 bond breaks in DMF. This detailed study of the simplest molecule with the peptide linkage contributes to our understanding of electron impact on peptide-like molecules.

Molecular evolution of the Slc15 family and its response to waterborne copper and mercury exposure in tilapia

The solute carrier 15 family (Slc15), also called oligopeptide transporter family (Pept), was well-known for its role in the cellular uptake of di/tripeptides and peptide-like molecules. Our understanding of Slc15 family has already been enlarged since the rapid increasing of genome information; however, efforts are still expected to reveal the diversification of the family in an evolutionary manner. In the study, the sequence information were collected and analyzed through eleven eukaryotic organism representatives, especially in fish species. Gene expansion was observed through the evolution of the family. Further study was carried out with the representative species-Nile tilapia (Oreochromis niloticus). Tissue expression profiles were compared among members of the Slc15 family. Generally, they were all highly expressed both in the intestine and stomach, however, different members possessed its special tissue expression pattern. The mRNA levels of all the members (except Slc15a4) decreased after fasting while refeeding could restore the expression level. The recovery ability was impaired after exposure to environmental relevant concentration of copper (Cu2+, 160nmol/L). By contrast, mercury (Hg2+, 25nmol/L) did not exert significant impact on the recovery ability.

Improving the representation of peptide‐like inhibitor and antibiotic molecules in the Protein Data Bank

With the accumulation of a large number and variety of molecules in the Protein Data Bank (PDB) comes the need on occasion to review and improve their representation. The Worldwide PDB (wwPDB) partners have periodically updated various aspects of structural data representation to improve the integrity and consistency of the archive. The remediation effort described here was focused on improving the representation of peptide-like inhibitor and antibiotic molecules so that they can be easily identified and analyzed. Peptide-like inhibitors or antibiotics were identified in over 1000 PDB entries, systematically reviewed and represented either as peptides with polymer sequence or as single components. For the majority of the single-component molecules, their peptide-like composition was captured in a new representation, called the subcomponent sequence. A novel concept called “group” was developed for representing complex peptide-like antibiotics and inhibitors that are composed of multiple polymer and nonpolymer components. In addition, a reference dictionary was developed with detailed information about these peptide-like molecules to aid in their annotation, identification and analysis. Based on the experience gained in this remediation, guidelines, procedures, and tools were developed to annotate new depositions containing peptide-like inhibitors and antibiotics accurately and consistently. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 659–668, 2014.

Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT.

Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like molecules, using the inwardly directed H(+) gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug molecules such as β-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H(+) binding remains unanswered. Here we report the high-resolution crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochemical, and computational analyses suggested a mechanism for H(+)-coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.