MAMMALIAN HETEROGENEOUS RIBONUCLEOPROTEIN-A1 AND ITS CONSTITUENT DOMAINS - NUCLEIC-ACID INTERACTION, STRUCTURAL STABILITY AND SELF-ASSOCIATION
MAMMALIAN HETEROGENEOUS RIBONUCLEOPROTEIN-A1 AND ITS CONSTITUENT DOMAINS - NUCLEIC-ACID INTERACTION, STRUCTURAL STABILITY AND SELF-ASSOCIATION
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DOI：
10.1006/jmbi.1993.1093
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发表时间：
1993-02-20
影响因子：
5.6
通讯作者：
KARPEL, RL
中科院分区：
文献类型：
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作者：
CASASFINET, JR;SMITH, JD;KARPEL, RL
为了进一步了解真核异质性核糖核蛋白A1的结构与功能关系，特别是它的多个核酸相互作用结构域，我们研究了球状N结构域（UP 1）和序列重复的柔性C结构域的核酸结合特性，UP 1的热变性以及结合多核苷酸的伴随效应，以及全长蛋白质的自缔合特性。利用蛋白质色氨酸荧光作为探针，多核苷酸结合显示出稳定UP 1对热解折叠。UP 1-聚（胸苷酸）复合物的变性概况是双相的，这表明UP 1的两个子域的展开可以独立地发生。这与先前提出的结构一致，其中两个UP 1亚结构域中只有一个结合核酸。UP 1的亚结构域可以通过A1的受控蛋白水解来制备，进一步表明A1内的这两个球状片段通过暴露的柔性连接来连接。UP 1的圆二色性测量证实了以前的数据，这部分A1结合单链核酸非合作。UP 1清楚地显示出对具有2′-OH的单链核酸的偏好，因为其对poly（U）的亲和力是poly（dU）的三倍，并且是poly（dU）的五倍通过制备合成的肽聚合物进一步检查C-结构域的核酸相互作用性质（Mr约12，000），其含有16个残基序列的约7个重复，GNFGGGRGNYGGSRG，其又包含C-末端共有序列GN（F/Y）GG（G/S）RG的两个拷贝。该序列的聚合物对荧光多聚核糖核苷酸聚（乙烯腺苷酸）表现出显著的亲和力，在≤ 0·2 M-Na+下化学计量结合。复合物的形成伴随着聚集体形成的增加，如散射的外观所示。为了进行比较，通过McGhee和von Hippel的线性合作模型分析数据，尽管该模型可能不能完全描述在这种情况下形成的蛋白质-核酸复合物。与UP 1结构域的非合作结合模式相反，C-聚合物表现出中等的合作性，与全长A1的情况相当。虽然加入足够的NaCl逆转了相互作用，但在0·8 M-NaCl下仍可以观察到S形结合等温线（加入足够的聚合物）。这表明非静电相互作用对结合的自由能有显著贡献。肽聚合物对单链和双链以及核糖核酸和脱氧核糖核酸没有表现出任何明显的特异性。该聚合物催化DNA链间退火，先前在全长A1和A1衍生的C-末端片段中观察到的活性。一个非重复的合成肽，只含有一个拷贝的16个残基的序列，既不显示核酸结合，也不退火活性。这表明这些特性需要共有序列的重复。在存在或不存在核酸的情况下观察到完整的A1的自缔合，并且可以通过其C-末端结构域中的疏水相互作用介导。
With a view toward further understanding the structure-function relationships of the eukaryotic heterogeneous ribonucleoprotein (hnRNP) A1, and in particular its multiplicity of nucleic acid-interactive domains, we have studied the nucleic acid binding properties of the globular N-domain (UP1) and sequence-repetitive, flexible C-domain, the thermal denaturation of UP1 and the concomitant effects of binding polynucleotide,and the self-associative properties of the full-length protein. Utilizing protein tryptophan fluorescence as a probe, polynucleotide binding was shown to stabilize UP1 against thermal unfolding. The denaturation profile of UP1-poly(thymidylic acid)complexes was biphasic, suggesting that unfolding of the two subdomains of UP1 can occur independently. This is in agreement with a previously proposed structure in which only one of the two UP1 subdomains binds the nucleic acid. The subdomains of UP1 can be prepared by controlled proteolysis of A1, further indicating that these two globular segments within A1 are connected by an exposed, flexible linkage. Circular dichroism measurements on UP1 confirm previous data that this portion of A1 binds single-stranded nucleic acids non-co-operatively. UP1 clearly shows a preference for single-stranded nucleic acids with a 2′-OH, since its affinity for poly(U) is three times higher than for poly(dU), and five times higher than its affinity for poly(2′-OCH3U).The nucleic acid-interactive properties of the C-domain were further examined by preparing a synthetic peptide polymer (Mr≈ 12,000) containing about seven repeats of a 16-residue sequence, GNFGGGRGGNYGGSRG, which in turn comprises two copies of the C-terminal consensus, GN(F/Y)GG(G/S)RG. The polymer of this sequence exhibited significant affinity for the fluorescent polyribonucleotide, poly(ethenoadenylic acid), binding stoichiometrically at ≤ 0·2 M-Na+. Complex formation was accompanied by an increase in aggregate formation, as indicated by the appearance of scattering. For purposes of comparison, the data were analyzedvia, the linear co-operative model of McGhee and von Hippel, though this model may not be fully descriptive of the protein-nucleic acid complex(es) formed in this case. In contrast to the non-co-operative binding mode of the UP1 domain, the C-polymer exhibited moderate co-operativity, comparable to that seen with full-length A1. Although addition of sufficient NaCl reversed the interaction, a sigmoidal binding isotherm could still be observe (with sufficient added polymer) at 0·8 M-NaCl. This suggests that non-electrostatic interactions contribute significantly to the free energy of binding. The peptide polymer did not exhibit any apparent specificity for single-strandedversusdouble-stranded and ribonucleicversusdeoxyriblonucleic acids. The polymer catalyzed DNA interstrand annealing, an activity previously observed in full-length A1 and A1-derived C-terminal fragment. A non-repetitive synthetic peptide, containing only one copy of the 16-residues sequence, showed neither nucleic acid binding nor annealing activity. This suggests that the repetition of the consensus sequence is required for theses properties. Self-association of intact A1 was observed in the presence or absence of nucleic acids, and may be mediated by hydrophobic interactions residing in its C-terminal domain.