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PH-Dependent conformational changes in proteins and their effect on experimental pK(a)s: the case of Nitrophorin 4.

ABSTRACT: The acid-base behavior of amino acids is an important subject of study due to their prominent role in enzyme catalysis, substrate binding and protein structure. Due to interactions with the protein environment, their pK(a)s can be shifted from their solution values and, if a protein has two stable conformations, it is possible for a residue to have different "microscopic", conformation-dependent pK(a) values. In those cases, interpretation of experimental measurements of the pK(a) is complicated by the coupling between pH, protonation state and protein conformation. We explored these issues using Nitrophorin 4 (NP4), a protein that releases NO in a pH sensitive manner. At pH 5.5 NP4 is in a closed conformation where NO is tightly bound, while at pH 7.5 Asp30 becomes deprotonated, causing the conformation to change to an open state from which NO can easily escape. Using constant pH molecular dynamics we found two distinct microscopic Asp30 pK(a)s: 8.5 in the closed structure and 4.3 in the open structure. Using a four-state model, we then related the obtained microscopic values to the experimentally observed "apparent" pK(a), obtaining a value of 6.5, in excellent agreement with experimental data. This value must be interpreted as the pH at which the closed to open population transition takes place. More generally, our results show that it is possible to relate microscopic structure dependent pKa values to experimentally observed ensemble dependent apparent pK(a)s and that the insight gained in the relatively simple case of NP4 can be useful in several more complex cases involving a pH dependent transition, of great biochemical interest.

SUBMITTER: Di Russo NV

PROVIDER: S-EPMC3486867 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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pH-Dependent conformational changes in proteins and their effect on experimental pK(a)s: the case of Nitrophorin 4.

Di Russo Natali V NV Estrin Dario A DA Martí Marcelo A MA Roitberg Adrian E AE

PLoS computational biology 20121101 11

The acid-base behavior of amino acids is an important subject of study due to their prominent role in enzyme catalysis, substrate binding and protein structure. Due to interactions with the protein environment, their pK(a)s can be shifted from their solution values and, if a protein has two stable conformations, it is possible for a residue to have different "microscopic", conformation-dependent pK(a) values. In those cases, interpretation of experimental measurements of the pK(a) is complicated ...[more]

PMID: 23133364

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Project description:Human bocavirus 1 (HBoV1) and HBoV2-4 infect children and immunocompromised individuals, resulting in respiratory and gastrointestinal infections, respectively. Using cryo-electron microscopy and image reconstruction, the HBoV2 capsid structure was determined to 2.7 Å resolution at pH 7.4 and compared to the previously determined HBoV1, HBoV3, and HBoV4 structures. Consistent with previous findings, surface variable region (VR) III of the capsid protein VP3, proposed as a host tissue-tropism determinant, was structurally similar among the gastrointestinal strains HBoV2-4, but differed from HBoV1 with its tropism for the respiratory tract. Towards understanding the entry and trafficking properties of these viruses, HBoV1 and HBoV2 were further analyzed as species representatives of the two HBoV tropisms. Their cell surface glycan-binding characteristics were analyzed, and capsid structures determined to 2.5-2.7 Å resolution at pH 5.5 and 2.6, conditions normally encountered during infection. The data showed that glycans with terminal sialic acid, galactose, GlcNAc or heparan sulfate moieties do not facilitate HBoV1 or HBoV2 cellular attachment. With respect to trafficking, conformational changes common to both viruses were observed at low pH conditions localized to the VP N-terminus under the 5-fold channel, in the surface loops VR-I and VR-V and specific side-chain residues such as cysteines and histidines. The 5-fold conformational movements provide insight into the potential mechanism of VP N-terminal dynamics during HBoV infection and side-chain modifications highlight pH-sensitive regions of the capsid.IMPORTANCE Human bocaviruses (HBoVs) are associated with disease in humans. However, the lack of an animal model and a versatile cell culture system to study their life cycle limits the ability to develop specific treatments or vaccines. This study presents the structure of HBoV2, at 2.7 Å resolution, determined for comparison to the existing HBoV1, HBoV3, and HBoV4 structures, to enable the molecular characterization of strain and genus-specific capsid features contributing to tissue tropism and antigenicity. Furthermore, HBoV1 and HBoV2 structures determined under acidic conditions provide insight into capsid changes associated with endosomal and gastrointestinal acidification. Structural rearrangements of the capsid VP N-terminus, at the base of the 5-fold channel, demonstrate a disordering of a "basket" motif as pH decreases. These observations begin to unravel the molecular mechanism of HBoV infection and provide information for control strategies.

Dataset Information

PH-Dependent conformational changes in proteins and their effect on experimental pK(a)s: the case of Nitrophorin 4.

Publications

pH-Dependent conformational changes in proteins and their effect on experimental pK(a)s: the case of Nitrophorin 4.

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