The Crystal Structures of Human S100A12 in Apo Form and in Complex with Zinc: New Insights into S100A12 Oligomerisation

Moroz, Olga V.; Blagova, E.V.; Wilkinson, Anthony J.; Wilson, K.S.; Bronstein, Igor B.

doi:10.1016/j.jmb.2009.06.004

Cited by 89 publications

(129 citation statements)

References 80 publications

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“…S100 family proteins are typically homodimers; however higher-order oligomers can be formed under specific conditions (20)(21)(22). As described by us previously, Ca 2þ -S100A4 dimers can assemble into a continuous superhelical arrangement due to the interaction of the C-terminal tail with the target peptide binding cleft of symmetry-related molecules (5).…”

Section: Resultsmentioning

confidence: 95%

“…Although the inactive tetramer has not been characterized structurally, the peptide is thought to induce the formation of a nonnatural tetramer (35). The recent characterization of S100B, S100A8/A9 and S100A12 as well-defined oligomers comprised of two to four S100 dimers (20)(21)(22), suggests that the propensity to form higher-order structures may be a common feature of S100 proteins. This characteristic is also shared by S100A4 as we and others demonstrated that S100A4 can form tetramers or higher-order oligomers in the absence of added compounds (5 and 36).…”

Section: Discussionmentioning

confidence: 99%

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Phenothiazines inhibit S100A4 function by inducing protein oligomerization

Malashkevich

Dulyaninova

Ramagopal

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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S100A4, a member of the S100 family of Ca 2þ -binding proteins, regulates carcinoma cell motility via interactions with myosin-IIA. Numerous studies indicate that S100A4 is not simply a marker for metastatic disease, but rather has a direct role in metastatic progression. These observations suggest that S100A4 is an excellent target for therapeutic intervention. Using a unique biosensorbased assay, trifluoperazine (TFP) was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction. To examine the interaction of S100A4 with TFP, we determined the 2.3 Å crystal structure of human Ca 2þ -S100A4 bound to TFP. Two TFP molecules bind within the hydrophobic target binding pocket of Ca 2þ -S100A4 with no significant conformational changes observed in the protein upon complex formation. NMR chemical shift perturbations are consistent with the crystal structure and demonstrate that TFP binds to the target binding cleft of S100A4 in solution. Remarkably, TFP binding results in the assembly of five Ca 2þ -S100A4/TFP dimers into a tightly packed pentameric ring. Within each pentamer most of the contacts between S100A4 dimers occurs through the TFP moieties. The Ca 2þ -S100A4/prochlorperazine (PCP) complex exhibits a similar pentameric assembly. Equilibrium sedimentation and cross-linking studies demonstrate the cooperative formation of a similarly sized S100A4/TFP oligomer in solution. Assays examining the ability of TFP to block S100A4-mediated disassembly of myosin-IIA filaments demonstrate that significant inhibition of S100A4 function occurs only at TFP concentrations that promote S100A4 oligomerization. Together these studies support a unique mode of inhibition in which phenothiazines disrupt the S100A4/ myosin-IIA interaction by sequestering S100A4 via small molecule-induced oligomerization.calcium | X-ray crystallography | NMR | small molecule inhibitor | metastasis T he S100 proteins, of which there are more than 20 members, are characterized by their solubility in 100% saturated ammonium sulfate (1 and 2). Each S100 family member contains two Ca 2þ -binding loops; a C-terminal "typical" EF-hand comprised of 12 residues and an N-terminal pseudo EF-hand consisting of 14 residues. The basic organization of the S100 proteins is a symmetric, antiparallel homodimer, in which the N-and C-terminal helices (helices 1 and 4) from each subunit interact to form a stable four helix bundle that serves as the dimer interface. Calcium binding to the C-terminal typical EF-hand significantly alters the angle between helices 3 and 4, which flank the C-terminal Ca 2þ -binding loop, and exposes a hydrophobic cleft that constitutes a binding surface for target proteins (3-5). Thus the S100 proteins operate as calcium-activated switches that bind and regulate the activity of diverse protein targets. S100 proteins are expressed in a tissue and cell specific manner. Elevated expression of individual family members is associated with a number of human pathologies, including cardiomyopathies, cancer, neurodegeneration, and in...

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Section: Resultsmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Phenothiazines inhibit S100A4 function by inducing protein oligomerization

Malashkevich

Dulyaninova

Ramagopal

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Based on sequence homology and the structural similarity among S100 proteins, the first predicted site (S1) is unique among S100 proteins and is composed of residues H17 and H27 from S100A8 and of H91 and H95 from S100A9 (13,19). The second predicted site (S2) involves residues H83 and H87 of S100A8 and of H20 and D30 of S100A9 and is similar to the canonical binding site observed in S100A7 and S100A12 (15,16). The binding of Zn and Mn to CP was completely eradicated in a variant in which all eight of these conserved residues were mutated (13).…”

mentioning

confidence: 81%

“…Two binding sites for Zn (and presumably other transition metals) have been identified at the dimer interface in crystal structures of Zn-bound S100B, S100A7, and S100A12 (15)(16)(17). Site-directed mutagenesis has been used to confirm that Zn and Mn are bound in the corresponding sites in CP (13).…”

mentioning

confidence: 99%

“…To distinguish the two sites, we engineered CP mutants that contain only one active transition metal-binding site using sequence conservation and structures of Zn-bound S100 proteins as a guide (13). Canonical transition metal-binding sites in S100 proteins are formed from a conserved HXXXH motif found at the C terminus of helix 4 of one subunit in combination with a histidine and aspartate residue from the other subunit (15,16). Whereas most S100 proteins are homodimers that have two identical transition metal-binding sites, CP is a heterodimer of S100A8 and S100A9 subunits, which results in two distinct binding sites (13,19).…”

mentioning

confidence: 99%

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Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens

Damo

Kehl-Fie

Sugitani

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

327

584

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The S100A8/S100A9 heterodimer calprotectin (CP) functions in the host response to pathogens through a mechanism termed "nutritional immunity." CP binds Mn 2+ and Zn 2+ with high affinity and starves bacteria of these essential nutrients. Combining biophysical, structural, and microbiological analysis, we identified the molecular basis of Mn 2+ sequestration. The asymmetry of the CP heterodimer creates a single Mn 2+ -binding site from six histidine residues, which distinguishes CP from all other Mn 2+ -binding proteins. Analysis of CP mutants with altered metal-binding properties revealed that, despite both Mn 2+ and Zn 2+ being essential metals, maximal growth inhibition of multiple bacterial pathogens requires Mn 2+ sequestration. These data establish the importance of Mn 2+ sequestration in defense against infection, explain the broad-spectrum antimicrobial activity of CP relative to other S100 proteins, and clarify the impact of metal depletion on the innate immune response to infection.bacterial pathogenesis | Staphylococcus aureus | antibiotic resistance | protein crystal structure | isothermal titration calorimetry B acterial pathogens are a significant threat to global public health. This threat is compounded by the fact that these organisms are rapidly becoming resistant to all relevant antimicrobials. Of particular note is the recent emergence of antibiotic-resistant strains of Staphylococcus aureus as a leading cause of bacterial infection in the United States (1) and arguably the most important threat to the public health of the developed world. Consequently, the identification of therapeutics to treat bacterial pathogens is paramount to our continued ability to limit this infectious threat.One promising area of potential therapeutic development involves targeting bacterial access to essential transition metals. This strategy is based on the fact that all bacterial pathogens require these nutrient metals to colonize their hosts (2-5). In vertebrates, the bacterial need for nutrient transition metals is counteracted by the sequestration of these metals by the host. This limitation of essential nutrients, termed "nutritional immunity," is a potent defense against infection (6). Although a variety of metals is required for microbial growth, studies of nutritional immunity have been primarily restricted to the struggle for iron (Fe) between host and pathogen (7-9).An innate immune factor, calprotectin (CP), is abundant in neutrophils and plays a key role in nutritional immunity. CP can be found at sites of infection in excess of 1 mg/mL and is required for the control of a number of medically relevant bacteria and fungi including S. aureus, Candida albicans, and Aspergillus fumigates (10-14). The antimicrobial activity of CP is due to the chelation of the essential nutrients Zn 2+ (Zn) and Mn 2+ (Mn), which results in bacterial metal starvation and is reversed by the addition of these metals in excess (11, 13). Moreover, CP-deficient mice have increased microbial burdens following systemic challenge, und...

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Calcium Regulates S100A12 Zinc Sequestration by Limiting Structural Variations

et al. 2020

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Antimicrobial proteins such as S100A12 and S100A8/A9 are highly expressed and secreted by neutrophils during infection and participate in human immune response by sequestering transition metals. At neutral pH, S100A12 sequesters Zn2+ with nanomolar affinity, which is further enhanced upon calcium binding. We investigated the pH dependence of human S100A12 zinc sequestration by using Co2+ as a surrogate. Apo‐S100A12 exhibits strong Co2+ binding between pH 7.0 and 10.0 that progressively diminishes as the pH is decreased to 5.3. Ca2+‐S100A12 can retain nanomolar Co2+ binding up to pH 5.7. NMR spectroscopic measurements revealed that calcium binding does not alter the side‐chain protonation of the Co2+/Zn2+ binding histidine residues. Instead, the calcium‐mediated modulation is achieved by restraining pH‐dependent conformational changes to EF loop 1, which contains Co2+/Zn2+ binding Asp25. This calcium‐induced enhancement of Co2+/Zn2+ binding might assist in the promotion of antimicrobial activities in humans by S100 proteins during neutrophil activation under subneutral pH conditions.

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The Crystal Structures of Human S100A12 in Apo Form and in Complex with Zinc: New Insights into S100A12 Oligomerisation

Cited by 89 publications

References 80 publications

Phenothiazines inhibit S100A4 function by inducing protein oligomerization

Phenothiazines inhibit S100A4 function by inducing protein oligomerization

Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens

Calcium Regulates S100A12 Zinc Sequestration by Limiting Structural Variations

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