Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

Setty, Thanuja Gangi; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

doi:10.1107/s139900471400830x

Cited by 51 publications

(41 citation statements)

References 28 publications

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“…3) led to a K d value, which is close to previously determined values using isothermal titration calorimetry (ITC) or Trp fluorescence quenching (5,6,23). This suggests that the PELDOR data can be quantitatively analyzed and, judged by the error calculated from the linear combination fitting of VcSiaP Q54R1/L173R1 time traces, it appears that as little as 3% of the closed conformation can be detected (Table S2).…”

Section: Discussionsupporting

confidence: 84%

“…Of course, it has to be determined for each particular case, which signal/noise is really needed to accurately distinguish the two states. However, the method might be advantageous for large K d values: for the R125A and R125K mutants, no Neu5Ac binding had previously been detected via ITC (23); in contract, the PELDOR experiment indicates weak binding. It should be noted that the PELDOR samples were flash-frozen.…”

Section: Discussionmentioning

confidence: 99%

“…As mentioned above, the R125-E184-H207 triad (henceforth referred to as ''triad'') is located in the hinge I and II regions of Neu5Ac transporter P domains, implicating a role in triggering the conformational change between substrate-free-(open) and -bound (closed) states of the protein (23). In a previous study, isothermal titration calorimetry was used to determine the Neu5Ac binding characteristics of triad mutants (23).…”

Section: Discussionmentioning

confidence: 99%

“…In Neu5Ac binding P domains, a group of three conserved, polar amino acids, R125, E184, and H207 were observed to form an intricate network of interactions (23). It has been proposed that these residues play an important role in the open-closed transition of P domains, also because they are close to two hinge regions in the structure (Fig.…”

Section: Mutational Analysis Of the Open-closed Transitionmentioning

confidence: 99%

“…It has been proposed that these residues play an important role in the open-closed transition of P domains, also because they are close to two hinge regions in the structure (Fig. 4 A) (23).…”

Section: Mutational Analysis Of the Open-closed Transitionmentioning

confidence: 99%

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PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

Glaenzer

Peter

Thomas

et al. 2017

Biophysical Journal

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The tripartite ATP-independent periplasmic (TRAP) transporters are a widespread class of membrane transporters in bacteria and archaea. Typical substrates for TRAP transporters are organic acids including the sialic acid N-acetylneuraminic acid. The substrate binding proteins (SBP) of TRAP transporters are the best studied component and are responsible for initial high-affinity substrate binding. To better understand the dynamics of the ligand binding process, pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy was applied to study the conformational changes in the N-acetylneuraminic acid-specific SBP VcSiaP. The protein is the SBP of VcSiaPQM, a sialic acid TRAP transporter from Vibrio cholerae. Spin-labeled double-cysteine mutants of VcSiaP were analyzed in the substrate-bound and -free state and the measured distances were compared to available crystal structures. The data were compatible with two clear states only, which are consistent with the open and closed forms seen in TRAP SBP crystal structures. Substrate titration experiments demonstrated the transition of the population from one state to the other with no other observed forms. Mutants of key residues involved in ligand binding and/or proposed to be involved in domain closure were produced and the corresponding PELDOR experiments reveal important insights into the open-closed transition. The results are in excellent agreement with previous in vivo sialylation experiments. The structure of the spin-labeled Q54R1/L173R1 R125A mutant was solved at 2.1 Å resolution, revealing no significant changes in the protein structure. Thus, the loss of domain closure appears to be solely due to loss of binding. In conclusion, these data are consistent with TRAP SBPs undergoing a simple two-state transition from an openunliganded to closed-liganded state during the transport cycle.

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Section: Discussionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Mutational Analysis Of the Open-closed Transitionmentioning

confidence: 99%

Section: Mutational Analysis Of the Open-closed Transitionmentioning

confidence: 99%

See 3 more Smart Citations

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

Glaenzer

Peter

Thomas

et al. 2017

Biophysical Journal

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Sialic Acid 4‐N‐Piperazine and Piperidine Derivatives Bind with High Affinity to the P. mirabilis Sialic Acid Sodium Solute Symporter

et al. 2022

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In search for novel antibacterial compounds, bacterial sialic acid uptake inhibition represents a promising strategy. Sialic acid plays a critical role for growth and colonisation of several pathogenic bacteria, and its uptake inhibition in bacteria was recently demonstrated to be a viable strategy by targeting the SiaT sodium solute symporters from Proteus mirabilis and Staphylococcus aureus. Here we report the design, synthesis and evaluation of potential sialic acid uptake inhibitors bearing 4-N-piperidine and piperazine moieties. The 4-N-derivatives were obtained via 4-N-functionalization with piperidine and piperazine nucleophiles in an efficient direct substitution of the 4-Oacetate of Neu5Ac. Evaluation for binding to bacterial transport proteins with nanoDSF and ITC revealed compounds possessing nanomolar affinity for the P. mirabilis SiaT symporter. Computational analyses indicate the engagement of a previously untargeted portion of the binding site.

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Quaternary variations in the structural assembly ofN‐acetylglucosamine‐6‐phosphate deacetylase fromPasteurella multocida

et al. 2020

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N-acetylglucosamine 6-phosphate deacetylase (NagA) catalyzes the conversion of Nacetylglucosamine-6-phosphate to glucosamine-6-phosphate in amino sugar catabolism. This conversion is an essential step in the catabolism of sialic acid in several pathogenic bacteria, including Pasteurella multocida, and thus NagA is identified as a potential drug target. Here, we report the unique structural features of NagA from P. multocida (PmNagA) resolved to 1.95Å. PmNagA displays an altered quaternary architecture with unique interface interactions compared to its close homolog, the Escherichia coli NagA (EcNagA). We confirmed that the altered quaternary structure is not a crystallographic artefact using single particle electron cryo-microscopy. Analysis of the determined crystal structure reveals a set of hot-spot residues involved in novel interactions at the dimer-dimer interface. PmNagA binds to one Zn 2+ ion in the active site and demonstrates kinetic parameters comparable to other bacterial homologs. Kinetic studies reveal that at high substrate concentrations (~10-fold the KM), the tetrameric PmNagA displays hysteresis similar to its distant neighbor, the dimeric Staphylococcus aureus NagA (SaNagA). Our findings provide key information on structural and functional properties of NagA in P. multocida that could be utilized to design novel antibacterials.

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Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

Abstract: Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding.

Cited by 51 publications

References 28 publications

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

PELDOR Spectroscopy Reveals Two Defined States of a Sialic Acid TRAP Transporter SBP in Solution

Sialic Acid 4‐N‐Piperazine and Piperidine Derivatives Bind with High Affinity to the P. mirabilis Sialic Acid Sodium Solute Symporter

Quaternary variations in the structural assembly ofN‐acetylglucosamine‐6‐phosphate deacetylase fromPasteurella multocida

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